Anti-cyanovirin antibody with an internal image of gp120, a method of use thereof, and a method of using a cyanovirin to induce an immune response to gp120

ABSTRACT

The present invention provides an anti-cyanovirin antibody with an internal image of gp120, a method of using an anti-cyanovirin antibody with an internal image of gp120 to induce an immune response to gp120 so as to prevent or treat a viral infection in an animal, and a method of using a cyanovirin to induce an immune response to gp120 so as to prevent or treat a viral infection in an animal.

CROSS-REFERENCE TO RELATED APPLICATION

This patent application is a continuation-in-part of U.S. patentapplication Ser. No. 08/969,249, filed Nov. 13, 19997 now U.S. Pat. No.5,998,587, which is a divisional of U.S. patent application Ser. No.08/638,610, filed Apr. 26, 19996 now U.S. Pat. No. 5,821,081, which is acontinuation-in-part of U.S. patent application Ser. No. 08/429,965,filed Apr. 27, 19995 now U.S. Pat. No. 5,843,882, all of which arehereby specifically incorporated by reference in their entireties.

TECHNICAL FIELD OF THE INVENTION

This invention relates to an anti-cyanovirin antibody with an internalimage of gp120, a method of using an anti-cyanovirin antibody with aninternal image of gp120 to induce an immune response to gp120, and amethod of using a cyanovirin to induce an immune response to gp120.

BACKGROUND OF THE INVENTION

Acquired immune deficiency syndrome (AIDS) is a fatal disease, reportedcases of which have increased dramatically within the past two decades.The virus that causes AIDS was first identified in 1983. It has beenknown by several names and acronyms. It is the third knownT-lymphotropic virus (HTLV-III), and it has the capacity to replicatewithin cells of the immune system, causing profound cell destruction.The AIDS virus is a retrovirus, a virus that uses reverse transcriptaseduring replication. This particular retrovirus also has been known aslymphadenopathy-associated virus (LAV), AIDS-related virus (ARV) and,presently, as human immunodeficiency virus (HIV). Two distinct familiesof HIV have been described to date, namely HIV-1 and HIV-2. The acronymHIV is used herein to refer generically to human immunodeficiencyviruses.

HIV exerts profound cytopathic effects on the CD4⁺ helper/inducerT-cells, thereby severely compromising the immune system. HIV infectionalso results in neurological deterioration and, ultimately, in death ofinfected individuals. Tens of millions of people are infected with HIVworldwide, and, without effective therapy, most of these are doomed todie. During the long latency, the period of time from initial infectionto the appearance of symptoms, or death, due to AIDS, infectedindividuals spread the infection further, by sexual contacts, exchangesof contaminated needles during i.v. drug abuse, transfusions of blood orblood products, or maternal transfer of HIV to a fetus or newborn. Thus,there is not only an urgent need for effective therapeutic agents toinhibit the progression of HIV disease in individuals already infected,but also for methods of prevention of the spread of HIV infection frominfected individuals to noninfected individuals. Indeed, the WorldHealth Organization (WHO) has assigned an urgent international priorityto the search for an effective anti-HIV prophylactic virucide to helpcurb the further expansion of the AIDS pandemic (Balter, Science 266,1312-1313, 1994; Merson, Science 260, 1266-1268, 1993; Taylor, J. NIHRes. 6, 26-27, 1994; Rosenberg et al., Sex. Transm. Dis. 20, 41-44,1993; Rosenberg, Am. J. Public Health 82, 1473-1478, 1992).

The field of viral therapeutics has developed in response to the needfor agents effective against retroviruses, especially HIV. There aremany ways in which an agent can exhibit anti-retroviral activity (see,e.g., DeClercq, Adv. Virus Res. 42, 1-55, 1993; DeClercq, J. Acquir.Immun. Def. Synd. 4, 207-218, 1991; Mitsuya et al., Science 249,1533-1544, 1990). Nucleoside derivatives, such as AZT, which inhibit theviral reverse transcriptase, are among the few clinically active agentsthat are currently available commercially for anti-HIV therapy. Althoughvery useful in some patients, the utility of AZT and related compoundsis limited by toxicity and insufficient therapeutic indices for fullyadequate therapy. Also, given more recent revelations of the dynamics ofHIV infection (Coffin, Science 267, 483-489, 1995; Cohen, Science 267,179, 1995; Perelson et al., Science 271, 1582-1586, 1996), it is nowincreasingly apparent that agents acting as early as possible in theviral replicative cycle are needed to inhibit infection of newlyproduced, uninfected immune cells generated in the body in response tothe virus-induced killing of infected cells. Also, it is essential toneutralize or inhibit new infectious virus produced by infected cells.

Infection of CD4⁺ cells by HIV-1 and related primate immunodeficiencyviruses begins with interaction of the respective viral envelopeglycoproteins (generically termed “gp120”) with the cell-surfacereceptor CD4, followed by fusion and entry (Sattentau, AIDS 2, 101-105,1988; Koenig et al., PNAS USA 86, 2443-2447, 1989). Productivelyinfected, virus-producing cells express gp120 at the cell surface;interaction of gp120 of infected cells with CD4 on uninfected cellsresults in formation of dysfunctional multicellular syncytia and furtherspread of viral infection (Freed et al., Bull. Inst. Pasteur 88, 73,1990). Thus, the gp120/CD4 interaction is a particularly attractivetarget for interruption of HIV infection and cytopathogenesis, either byprevention of initial virus-to-cell binding or by blockage ofcell-to-cell fusion (Capon et al., Ann. Rev. Immunol. 9, 649-678, 1991).Virus-free or “soluble” gp120 shed from virus or from infected cells invivo is also an important therapeutic target, since it may otherwisecontribute to noninfectious immunopathogenic processes throughout thebody, including the central nervous system (Capon et al., 1991, supra;Lipton, Nature 367, 113-114, 1994). Much vaccine research has focusedupon gp120; however, progress has been hampered by hypervariability ofthe gp120-neutralizing determinants and the consequent extremestrain-dependence of viral sensitivity to gp120-directed antibodies(Berzofsky, J. Acq. Immun. Def. Synd. 4, 451-459, 1991). Considerableeffort has been devoted to truncated, recombinant “CD4” proteins(“soluble CD4” or “sCD4”), which bind to gp120 and inhibit HIVinfectivity in vitro (Capon et al., 1991, supra; Schooley et al., Ann.Int. Med. 112, 247-253, 1990; Husson et al., J. Pediatr. 121, 627-633,1992). However, clinical isolates, in contrast to laboratory strains ofHIV, have proven highly resistant to neutralization by sCD4 (Orloffetal., AIDS Res. Hum. Retrovir. 11, 335-342, 1995; Moore et al., J. Virol.66, 235-243, 1992). Initial clinical trials of sCD4 (Schooley et al.,1990, supra; Husson et al., 1992, supra), and of sCD4-coupledimmunoglobulins (Langner et al., Arch. Virol. 130, 157-170, 1993), andlikewise of sCD4-coupled toxins designed to bind and destroyvirus-expressing cells (Davey et al., J. Infect. Dis. 170, 1180-1188,1994; Ramachandran et al., J. Infect. Dis. 170, 1009-1013, 1994), havebeen disappointing. Newer gene-therapy approaches to generating sCD4directly in vivo (Morgan et al., AIDS Res. Hum. Retrovir. 10, 1507-1515,1994) will likely suffer similar frustrations.

The development of a safe and effective vaccine is now considered to bethe single most important long-term goal of current research efforts.However, as pointed out by Varmus and Nathanson (Science 280, 1815,1998), this is a daunting, if not impossible, task. Many past effortsfocused on eliciting a neutralizing antibody response against theenvelope complex (reviewed in Burton, PNAS USA, 94, 10018-10023, 1997;see, also, Haynes, Lancet, 348, 933-937, 1996). Current strategies fordevelopment of an HIV-1 vaccine include live, attenuated virus,inactivated virus combined with an adjuvant, a subunit vaccine, e.g., arecombinant monomeric envelope protein or a peptide, a live vector-basedvaccine, and the use of DNA plasmids. However, each approach has itslimitations. For example, the use of live, attenuated virus poses therisk of eventual pathogenicity in vaccines. Use of inactivated viruscombined with an adjuvant results in anti-cellular, rather thananti-viral, antibodies. The absence of neutralizing antibodies presentsa problem for subunit vaccines. In fact, human vaccine trials usingmonomeric gp120 as an immunogen were disappointing at best (VanCott etal., J. Immunol., 155, 4100-4110, 1995; Haynes, Lancet, 348, 933-937,1996; Bolognesi et al., Nature, 391, 638-639, 1998; and Connor et al.,J. Virol., 72, 1552-1576, 1998). Immunogenicity limits the efficacy oflive vector-based vaccines. The use of DNA plasmids is limited bycurrent experience.

Anti-idiotypic antibodies, which carry an internal image of an epitopeof an antigen and which are designated as Ab2β antibodies, have beenshown to induce protective immunity in animals that intentionally havenot been exposed to the native antigen epitope (Poskitt et al., Vaccine,9, 792-796, 1991; and Greenspan et al., FASEB J., 7, 437-444, 1993).Furthermore, anti-idiotypic antibodies have been shown to have potentialfor immunotherapy of colorectal cancer and ovarian cancer (Denton etal., Int. J. Cancer, 57, 10-14, 1994; Madiyalakan et al., Hybridoma,14(2), 199-203 (1995); and Wagner et al., Hybridoma, 16(1), 33-40(1997)).

Therefore, new antiviral agents, such as anti-idiotypic antibodies, tobe used alone or in combination with AZT and/or other availableantiviral agents, are needed for effective antiviral therapy againstAIDS. New agents, which may be used to prevent HIV infection, also areimportant for prophylaxis. In both areas of need, the ideal new agentswould act as early as possible in the viral life cycle, be asvirus-specific as possible (i.e., attack a molecular target specific tothe virus but not to the infected or infectible animal host), render theintact virus noninfectious, prevent the death or dysfunction ofvirus-infected mammalian cells, prevent further production of virus frominfected cells, prevent spread of virus infection to uninfectedmammalian cells, be highly potent and active against the broadestpossible range of strains and isolates of HIV, be resistant todegradation under physiological and rigorous environmental conditions,and be readily and inexpensively produced on a large-scale.

The present invention seeks to provide such antiviral agents as well asmethods of using such antiviral agents to prevent or treat a viralinfection in an animal. These and other objects and advantages of thepresent invention, as well as additional inventive features, will becomeapparent from the description provided herein.

BRIEF SUMMARY OF THE INVENTION

The present invention provides an anti-cyanovirin antibody, which has aninternal image of gp120 of a primate immunodeficiency virus. Preferably,the antibody can compete with gp120 of a primate immunodeficiency virusfor binding to a cyanovirin. In this regard, the primateimmunodeficiency virus preferably is HIV-1 or HIV-2 and the cyanovirinpreferably comprises SEQ ID NO: 2.

The present invention also provides a method of preventing or treating aviral infection in an animal. The method comprises administering to theanimal an anti-cyanovirin antibody in an amount sufficient to induce inthe animal an immune response to a virus sufficient to prevent or treatan infection of the animal with the virus. The anti-cyanovirin antibodyhas an internal image of gp120 of a primate immunodeficiency virus.Preferably, the antibody can compete with gp120 of a primateimmunodeficiency virus for binding to a cyanovirin. In this regard, theprimate immunodeficiency virus preferably is HIV-1 or HIV-2 and thecyanovirin preferably comprises SEQ ID NO: 2.

Also provided by the present invention is another method of preventingor treating a viral infection in an animal. The method comprisesadministering to the animal a cyanovirin, which binds gp120 of a primateimmunodeficiency virus, in an amount sufficient to induce in the animalan anti-cyanovirin antibody in an amount sufficient to induce an immuneresponse to a virus sufficient to treat or prevent an infection of theanimal with the virus. Preferably, the anti-cyanovirin antibody cancompete with gp120 of a primate immumodeficiency virus for binding to acyanovirin. In this regard, the primate immunodeficiency viruspreferably is HIV-1 or HIV2 and the cyanovirin preferably comprises SEQID NO: 2.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph of OD (206 nrm) versus time (min), which representsan HPLC chromatogram of nonreduced cyanovirin-N.

FIG. 1B is a bar-graph of maximum dilution for 50% protection versusHPLC fraction, which illustrates the maximum dilution of each HPLCfraction that provided 50% protection from the cytopathic effects of HIVinfection for the nonreduced cyanovirin-N HPLC fractions.

FIG. 1C is an SDS-polyacrylamide gel electrophoretogram of nonreducedcyanovirin-N HPLC fractions.

FIG. 1D is a graph of OD (206 nrm) versus time (min), which representsan HPLC chromatogram of reduced cyanovirin-N.

FIG. 1E is a bar graph of maximum dilution for 50% protection versusHPLC dilution, which illustrates the maximum dilution of each fractionthat provided 50% protection from the cytopathic effects of HIVinfection for the reduced cyanovirin-N HPLC fractions.

FIG. 1F is an SDS-polyacrylamide gel electrophoretogram of reducedcyanovirin-N HPLC fractions.

FIG. 2 shows an example of a DNA sequence encoding a syntheticcyanovirin gene.

FIG. 3 illustrates a site-directed mutagenesis maneuver used toeliminate codons for a FLAG octapeptide and a Hind III restriction sitefrom the sequence of FIG. 2.

FIG. 4 shows a typical HPLC chromatogram from the purification ofrecombinant native cyanovirin-N.

FIG. 5A is a graph of % control versus cyanovirin-N concentration (nM),which illustrates the antiviral activity of native cyanovirin-N fromNostoc ellipsosporum.

FIG. 5B is a graph of % control versus cyanovirin-N concentration (nM),which illustrates the antiviral activity of recombinant cyanovirin-N.

FIG. 5C is a graph of % control versus cyanovirin-N concentration (nM),which illustrates the antiviral activity of recombinantFLAG-cyanovirin-N.

FIG. 6A is a graph of % control versus cyanovirin-N concentration (nM),which depicts the relative numbers of viable CEM-SS cells infected withHIV-1 in a BCECF assay.

FIG. 6B is a graph of % control versus cyanovirin-N concentration (nM),which depicts the relative DNA contents of CEM-SS cell cultures infectedwith HIV-1.

FIG. 6C is a graph of % control versus cyanovirin-N concentration (riM),which depicts the relative numbers of viable CEM-SS cells infected withHIV-1 in an XTT assay.

FIG. 6D is a graph of % control versus cyanovirin-N concentration (nM),which depicts the effect of a range of concentrations of cyanovirin-Nupon indices of infectious virus or viral replication.

FIG. 7 is a graph of % uninfected control versus time-of-addition (hr),which shows the results of delayed-addition studies of cyanovirin-N,showing anti-HIV activity in CEM-SS cells infected with HIV-1_(RF).

FIG. 8A is a graph of OD (450 nm) versus cyanovirin-N concentration(μg/ml), which illustrates cyanovirin/gp120 interactions defining gp120as a principal molecular target of cyanovirins.

FIG. 8B is a dot-blot of the binding of cyanovirin-N and a gp120-HRPconjugate, which shows that cyanovirin-N specifically bound ahorseradish peroxidase conjugate of gp120 (gp120-HRP) in aconcentration-dependent manner.

FIG. 9 schematically illustrates a DNA coding sequence comprising aFLAG-cyanovirin-N coding sequence coupled to a Pseudomonas exotoxincoding sequence.

FIG. 10 is a graph of OD (450 nM) versus PPE concentration (nM), whichillustrates selective killing of viral gp120-expressing (H9/IIIB) cellsby a FLAG-cyanovirin-N/Pseudomonas exotoxin protein conjugate (PPE).

FIG. 11 is a western-blot from an SDS-polyacrylamide gelelctrophoretogram of lysed COS-7 cells which had been engineered andtransformed to express a FLAG-cyanovirin-N; detection was by ananti-FLAG antibody.

FIG. 12 is a western-blot from an SDS-polyacrylamide gelelectrophoretogram of secreted products, digested with peptideN4-(N-acetyl-β-glucosaminyl) asparagine amidase, from Pichia pastorisengineered and transformed to produce a cyanovirin; detection was by ananti-cyanovirin-N polyclonal antibody.

FIG. 13 is an SDS-polyacrylamide gel electrophoretogram (A) and awestern-blot (B) from a whole-cell lysate from E. coli engineered toproduce cyanovirin-N; detection was by an anti-cyanovirin-N polyclonalantibody.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is predicated, at least in part, on theobservation that certain extracts from cultured cyanobacteria(blue-green algae) exhibited antiviral activity in an anti-HIV screen.The anti-HIV screen was conceived in 1986 (by M. R. Boyd of the NationalInstitutes of Health) and has been developed and operated at the U.S.National Cancer Institute (NCI) since 1988 (see Boyd, in AIDS, EtiologyDiagnosis, Treatment and Prevention, DeVita et al., eds., Philadelphia:Lippincott, 1988, pp. 305-317).

Cyanobacteria (blue-green algae) were specifically chosen for anti-HIVscreening because they had been known to produce a wide variety ofstructurally unique and biologically active non-nitrogenous and aminoacid-derived natural products (Faulkner, Nat. Prod. Rep. 11, 355-394,1994; Glombitza et al., in Algal and Cyanobacterial Biotechnology,Cresswell, R. C., et al. eds., 1989, pp. 211-218). These photosyntheticprocaryotic organisms are significant producers of cyclic and linearpeptides (molecular weight generally <3 kDa), which often exhibithepatotoxic or antimicrobial properties (Okino et al., Tetrahedron Lett.34, 501-504, 1993; Krishnamurthy et al., PNAS USA 86, 770-774, 1989;Sivonen et al., Chem. Res. Toxicol. 5, 464-469, 1992; Carter et al., J.Org. Chem. 49, 236-241, 1984; Frankmolle et al., J. Antibiot. 45,1451-1457, 1992). Sequencing studies of higher molecular weightcyanobacterial proteins have generally focused on those associated withprimary metabolic processes or ones that can serve as phylogeneticmarkers (Suter et al., FEBS Lett. 217, 279-282, 1987; Rumbeli et al.,FEBS Lett. 221, 1-2, 1987; Swanson et al., J. Biol. Chem. 267,16146-16154, 1992; Michalowski et al., Nucleic Acids Res. 18, 2186,1990; Sherman et al., in The Cyanobacteria, Fay et al., eds., Elsevier:New York, 1987, pp. 1-33; Rogers, in The Cyanobacteria, Fay et al.,eds., Elsevier: New York, 1987, pp. 35-67). In general, proteins withantiviral properties have not been associated with cyanobacterialsources.

The cyanobacterial extract leading to the present invention was amongmany thousands of different extracts initially selected randomly andtested blindly in the anti-HIV screen described above. A number of theseextracts had been determined preliminarily to show anti-HIV activity inthe NCI screen (Patterson et al., J. Phycol. 29, 125-130, 1993). Fromthis group, an aqueous extract from Nostoc ellipsosporum, which had beenprepared as described (Patterson, 1993, sura) and which showed anunusually high anti-HIV potency and in vitro “therapeutic index” in theNCI primary screen, was selected for detailed investigation. A specificbioassay-guided strategy was used to isolate and purify a homogenousprotein highly active against HIV.

In the bioassay-guided strategy, initial selection of the extract forfractionation, as well as the decisions concerning the overall chemicalisolation method to be applied, and the nature of the individual stepstherein, were determined by interpretation of biological testing data.The anti-HIV screening assay (see, e.g., Boyd, 1988, supra; Weislow etal., J. Natl. Cancer Inst. 81, 577-586, 1989), which was used to guidethe isolation and purification process, measures the degree ofprotection of human T-lymphoblastoid cells from the cytopathic effectsof HIV. Fractions of the extract of interest are prepared using avariety of chemical means and are tested blindly in the primary screen.Active fractions are separated further, and the resulting subfractionsare likewise tested blindly in the screen. This process is repeated asmany times as necessary in order to obtain the active compound(s), i.e.,antiviral fraction(s) representing pure compound(s), which then can besubjected to detailed chemical analysis and structural elucidation.

Using this strategy, aqueous extracts of Nostoc ellipsosporum werediscovered to contain an antiviral protein. It should be noted that theterm “protein” as used herein to describe the present invention is notrestricted to an amino acid sequence of any particular length andincludes molecules comprising 100 or more amino acids, as well asmolecules comprising less than 100 amino acids (which are sometimesreferred to as “peptides”).

In view of the above, an isolated and purified antiviral protein fromNostoc ellipsosporum, specifically an isolated and purified antiviralprotein known as cyanovirinN, is provided. Other cyanovirins are alsoprovided. The term “cyanovirin” is used herein to generically refer to anative antiviral protein isolated from Nostoc ellipsosporum (“nativecyanovirin”) and any functionally equivalent protein or derivativethereof. Cyanovirins and functionally equivalent proteins andderivatives thereof as described herein can inactivate a wide range ofHIV strains and isolates.

Such a functionally equivalent protein or derivative thereof (a)contains a sequence of at least nine (preferably at least twenty, morepreferably at least thirty, and most preferably at least fifty) aminoacids directly homologous with (preferably the same as) any subsequenceof nine contiguous amino acids contained within a native cyanovirin(especially cyanovirin-N), and (b) is antiviral, in particular capableof specifically binding to a virus, more specifically a primateimmunodeficiency virus, more specifically HIV-1, HIV-2, or SIV, or to aninfected host cell expressing one or more viral antigen(s), morespecifically an envelope glycoprotein, such as gp120, of the respectivevirus. In addition, such a functionally equivalent protein or derivativethereof can comprise the amino acid sequence of a native cyanovirin,particularly cyanovirin-N (see SEQ ID NO:2), in which 1-20, preferably1-10, more preferably 1, 2, 3, 4, or 5, and most preferably 1 or 2,amino acids have been removed from one or both ends, preferably fromonly one end, and most preferably from the amino-terminal end, of thenative cyanovirin.

The cyanovirin preferably comprises an amino acid sequence that issubstantially homologous to that of an antiviral protein from Nostocellipsosporum, specifically a native cyanovirin, particularlycyanovirin-N. In the context of the cyanovirins, the term “substantiallyhomologous” means sufficient homology to render the cyanovirinantiviral, preferably with antiviral activity characteristic of anantiviral protein isolated from Nostoc ellipsosporum. There preferablyexists at least about 50% homology, more preferably at least about 75%homology, and most preferably at least about 90% homology.

Thus, also provided is an isolated and purified protein encoded by anucleic acid molecule comprising a coding sequence for a cyanovirin,such as particularly an isolated and purified protein encoded by anucleic acid molecule comprising a sequence of SEQ ID NO:1, a nucleicacid molecule comprising a sequence of SEQ ID NO:3, a nucleic acidmolecule encoding an amino acid sequence of SEQ ID NO:2, or a nucleicacid molecule encoding an amino acid sequence of SEQ ID NO:4.

Likewise, a cyanovirin conjugate, which comprises a cyanovirin coupledto one or more selected effector molecule(s), such as a toxin orimmunological reagent, is provided. The term “immunological reagent” isused herein to refer to an antibody, an immunoglobulin, and animmunological recognition element. An immunological recognition elementis an element, such as a peptide, e.g., the FLAG sequence of therecombinant cyanovirin-FLAG fusion protein, which facilitates, throughimmunological recognition, isolation and/or purification and/or analysisof the protein to which it is attached. A cyanovirin fusion protein is atype of cyanovirin conjugate, wherein a cyanovirin is coupled to one ormore other protein(s) having any desired properties or effectorfunctions, such as cytotoxic or immunological properties, or otherdesired properties, such as to facilitate isolation, purification, oranalysis of the fusion protein.

Also provided is a method of obtaining a cyanovirin from Nostocellipsosporum. The method comprises (a) identifying an extract of Nostocellipsosporum containing antiviral activity, (b) optionally removinghigh molecular weight biopolymers from the extract, (c) antiviralbioassay-guided fractionating the extract to obtain a partially purifiedextract of cyanovirin, and (d) further purifying the partially purifiedextract by reverse-phase HPLC to obtain a cyanovirin (see Example 1).The method preferably involves the use of ethanol to remove highmolecular weight biopolymers from the extract and the use of an anti-HIVbioassay to guide fractionation of the extract.

The isolated and purified cyanovirin, such as cyanovirin-N (CV-N), canbe subjected to conventional procedures typically used to determine theamino acid sequence of a given pure protein. Thus, the cyanovirin can besequenced by N-terminal Edman degradation of intact protein andoverlapping peptide fragments generated by endoproteinase digestion.Amino acid analysis desirably will be in agreement with the deducedsequence. Similarly, ESI mass spectrometry of reduced, HPLC-purifiedcyanovirin-N desirably will show a molecular ion value consistent withthe calculated value.

These studies indicated that cyanovirin-N from Nostoc ellipsosporumcomprises a unique sequence of 101 amino acids having little or nosignificant homology to previously described proteins or transcriptionproducts of known nucleotide sequences. No more than eight contiguousamino acids from cyanovirin are found in any amino acid sequences fromknown proteins, nor are there any known proteins from any sourcecontaining greater than 13% sequence homology with cyanovirin-N. Giventhe chemically deduced amino acid sequence of cyanovirin-N, acorresponding recombinant cyanovirin-N (r-cyanovirin-N, or r-CV-N) wascreated and used to definitively establish that the deduced amino acidsequence is, indeed, active against viruses, such as HIV (see Examples2-5).

Also provided are an isolated and purified nucleic acid molecule and asynthetic nucleic acid molecule, which comprise a coding sequence for acyanovirin (particularly a native cyanovirin, especially cyanovirin-N).Such nucleic acid molecules include an isolated and purified nucleicacid molecule comprising a sequence of SEQ ID NO:1, an isolated andpurified nucleic acid molecule comprising a sequence of SEQ ID NO:3, anisolated and purified nucleic acid molecule encoding an amino acidsequence of SEQ ID NO:2, an isolated and purified nucleic acid moleculeencoding an amino acid sequence of SEQ ID NO:4, and a nucleic acidmolecule that is substantially homologous to any one or more of theaforementioned nucleic acid molecules. In the context of the nucleicacid molecule of the present invention, the term “substantiallyhomologous” means sufficient homology to render the protein encoded bythe nucleic acid molecule antiviral, preferably with antiviral activitycharacteristic of an antiviral protein isolated from Nostocellipsosporum. There preferably exists at least about 50% homology, morepreferably at least about 75% homology, and most preferably at leastabout 90% homology.

The nucleic acid molecule desirably comprises a nucleic acid sequenceencoding at least nine (preferably at least twenty, more preferably atleast thirty, and most preferably at least fifty) contiguous amino acidsof the amino acid sequence of SEQ ID NO:2. The present inventive nucleicacid molecule also desirably comprises a nucleic acid sequence encodinga protein comprising the amino acid sequence of a native cyanovirin,particularly cyanovirin-N, in which 1-20, preferably 1-10, morepreferably 1, 2, 3, 4, or 5, and most preferably 1 or 2, amino acidshave been removed from one or both ends, preferably from only one end,and most preferably from the amino-terminal end, of the nativecyanovirin.

In view of the above, it will be apparent to one skilled in the art thata partial cyanovirin-N gene codon sequence will likely suffice to codefor a fully functional, i.e., antiviral, such as anti-HIV, cyanovirin. Aminimum essential DNA coding sequence(s) for a functional cyanovirin canreadily be determined by one skilled in the art, for example, bysynthesis and evaluation of sub-sequences comprising the nativecyanovirin, and by site-directed mutagenesis studies of the cyanovirin-NDNA coding sequence.

Using an appropriate DNA coding sequence, a recombinant cyanovirin canbe made by genetic engineering techniques (see, e.g., for generalbackground, Nicholl, in An Introduction to Genetic Engineering,Cambridge University Press: Cambridge, 1994, pp. 1-5 & 127-130;Steinberg et al., in Recombinant DNA Technology Concepts and BiomedicalApplications, Prentice Hall: Englewood Cliffs, N.J., 1993, pp. 81-124 &150-162; Sofer in Introduction to Genetic Engineering,Butterworth-Heinemann, Stoneham, Mass., 1991, pp. 1-21 & 103-126; Old etal., in Principles of Gene Manipulation, Blackwell ScientificPublishers: London, 1992, pp. 1-3 & 108-221; Emtage, in Delivery Systemsfor Peptide Drugs, Davis et al., eds., Plenum Press: New York, 1986, pp.23-33). For example, a Nostoc ellipsosporum gene or cDNA encoding acyanovirin can be identified and subcloned. The gene or cDNA can then beincorporated into an appropriate expression vector and delivered into anappropriate protein-synthesizing organism (e.g., E. coli, S. cerevisiae,P. pastoris, or other bacterial, yeast, insect, or mammalian cell),where the gene, under the control of an endogenous or exogenouspromoter, can be appropriately transcribed and translated. Suchexpression vectors (including, but not limited to, phage, cosmid, viral,and plasmid vectors) are known to those skilled in the art, as arereagents and techniques appropriate for gene transfer (e.g.,transfection, electroporation, transduction, micro-injection,transformation, etc.). Subsequently, the recombinantly produced proteincan be isolated and purified using standard techniques known in the art(e.g., chromatography, centrifugation, differential solubility,isoelectric focusing, etc.), and assayed for antiviral activity.

Alternatively, a native cyanovirin can be obtained from Nostocellipsosporum by non-recombinant methods (see, e.g., Example 1 andforegoing discussion) and sequenced by conventional techniques. Thesequence can then be used to synthesize the corresponding DNA, which canbe subcloned into an appropriate expression vector and delivered into aprotein-producing cell for en mass recombinant production of the desiredprotein.

In this regard, a vector comprising a nucleic acid molecule encoding acyanovirin, e.g., a DNA sequence such as a Nostoc ellipsosporum genesequence for cyanovirin, a cDNA encoding a cyanovirin, or a syntheticDNA sequence encoding a cyanovirin, is provided. Also provided is a hostcell comprising such a nucleic acid molecule or vector, as well as amethod of using such a host cell to produce a cyanovirin.

The DNA, whether isolated and purified or synthetic, or cDNA encoding acyanovirin can encode for either the entire cyanovirin or a portionthereof (desirably an antivirally active portion thereof). Where the DNAor cDNA does not comprise the entire coding sequence of the nativecyanovirin, the DNA or cDNA can be subcloned as part of a gene fusion.In a transcriptional gene fusion, the DNA or cDNA will contain its owncontrol sequence directing appropriate production of protein (e.g.,ribosome binding site, translation initiation codon, etc.), and thetranscriptional control sequences (e.g., promoter elements and/orenhancers) will be provided by the vector. In a translational genefusion, transcriptional control sequences as well as at least some ofthe translational control sequences (i.e., the translational initiationcodon) will be provided by the vector. In the case of a translationalgene fusion, a chimeric protein will be produced.

Genes also can be constructed for specific fusion proteins containing afunctional cyanovirin component plus a fusion component conferringadditional desired attribute(s) to the composite protein. For example, afusion sequence for a toxin or immunological reagent, as defined above,can be added to facilitate purification and analysis of the functionalprotein (e.g., such as the FLAG-cyanovirin-N fusion protein described inExamples 2-5).

Genes can be specifically constructed to code for fusion proteins, whichcontain a cyanovirin coupled to an effector protein, such as a toxin orimmunological reagent, for specific targeting to viral-infected, e.g.,HIV and/or HIV-infected, cells. In these instances, the cyanovirinmoiety serves not only as a neutralizing agent but also as a targetingagent to direct the effector activities of these molecules selectivelyagainst a given virus, such as HIV. Thus, for example, a therapeuticagent can be obtained by combining the HIV-targeting function of afunctional cyanovirin with a toxin aimed at neutralizing infectiousvirus and/or by destroying cells producing infectious virus, such asHIV. Similarly, a therapeutic agent can be obtained, which combines theviraltargeting function of a cyanovirin with the multivalency andeffector functions of various immunoglobulin subclasses. Example 6further illustrates the viral-targeting, specifically gp120-targeting,properties of a cyanovirin.

Similar rationales underlie extensive developmental therapeutic effortsexploiting the HIV gp120-targeting properties of sCD4. For example,sCD4-toxin conjugates have been prepared in which sCD4 is coupled to aPseudomonas exotoxin component (Chaudhary et al., in The HumanRetrovirus, Gallo et al., eds., Academic Press: San Diego, 1991, pp.379-387; Chaudhary et al., Nature 335, 369-372, 1988), a diphtheriatoxin component (Aullo et al., EMBO J. 11, 575-583, 1992), or a ricinA-chain component (Till et al., Science 242, 1166-1167, 1988). Likewise,sCD4-immunoglobulin conjugates have been prepared in attempts todecrease the rate of in vivo clearance of functional sCD4 activity, toenhance placental transfer, and to effect a targeted recruitment ofimmunological mechanisms of pathogen elimination, such as phagocyticengulfment and killing by antibody-dependent cell-mediated cytotoxicity,to kill and/or remove HIV-infected cells and virus (Capon et al., Nature337, 525-531, 1989; Traunecker et al., Nature 339, 68-70, 1989; Langneret al., 1993, supra). While such CD4-immunoglobulin conjugates(sometimes called “immunoadhesins”) have, indeed, shown advantageouspharmacokinetic and distributional attributes in vivo, and anti-HIVeffects in vitro, clinical results have been discouraging (Schooley etal., 1990, supra; Husson et al., 1992, supra; Langner et al., 1993,supra). This is not surprising since clinical isolates of HIV, asopposed to laboratory strains, are highly resistant to binding andneutralization by sCD4 (Orloff et al., 1995, supra; Moore et al., 1992,supra). Therefore, the extraordinarily broad antiviral activity andtargeting properties of a functional cyanovirin to viruses, e.g.,primate retroviruses, in general, and clinical and laboratory strains,in particular (see, e.g., Example 7), are especially advantageous forcombining with toxins, immnunoglobulins, and other selected effectorproteins.

Viral-targeted conjugates can be prepared either by genetic engineeringtechniques (see, for example, Chaudhary et al., 1988, supra) or bychemical coupling of the targeting component with an effector component.The most feasible or appropriate technique to be used to construct agiven cyanovirin conjugate or fusion protein will be selected based uponconsideration of the characteristics of the particular effector moleculeselected for coupling to a cyanovirin. For example, with a selectednon-proteinaceous effector molecule, chemical coupling, rather thangenetic engineering techniques, represents the most feasible option forcreating the desired cyanovirin conjugate.

Accordingly, nucleic acid molecules encoding cyanovirin fusion proteins,in addition to the cyanovirin fusion proteins themselves, are provided.In particular, a nucleic acid molecule comprising SEQ ID NO:3 andsubstantially homologous sequences thereto are provided. A vectorcomprising a nucleic acid sequence encoding a cyanovirin fusion proteinis provided as is a method of obtaining a cyanovirin fusion protein byexpression of the vector encoding a cyanovirin fusion protein in aprotein-synthesizing organism as described above.

Further provided is an isolated and purified nucleic acid moleculecomprising a first nucleic acid sequence that encodes a protein of thepresent invention, e.g., a cyanovirin coding sequence, such as one ofthe aforementioned nucleic acids, coupled to a second nucleic acidencoding an effector protein, such as a toxin or immunological reagentas described above. Also provided is an isolated and purified proteinencoded by such a nucleic acid molecule.

The coupled molecule (conjugate) desirably targets a virus, morepreferably HIV, and most preferably glycoprotein gp120. The coupling canbe effected at the DNA level or by chemical coupling as described above.For example, a cyanovirin-effector protein conjugate of the presentinvention can be obtained by (a) selecting a desired effector protein,(b) synthesizing a composite DNA coding sequence comprising a first DNAcoding sequence comprising one of the aforementioned nucleic acidsequences, which codes for a functional cyanovirin, coupled to a secondDNA coding sequence for an effector protein, e.g., a toxin orimmunological reagent, (c) expressing the composite DNA coding sequencein an appropriate protein-synthesizing organism, and (d) purifying thedesired fusion protein to substantially pure form. Alternatively, acyanovirin-effector molecule conjugate can be obtained by (a) selectinga desired effector molecule and a cyanovirin or cyanovirin fusionprotein, (b) chemically coupling the cyanovirin or cyanovirin fusionprotein to the effector molecule, and (c) isolating the desiredcyanovirin-effector molecule conjugate in substantially pure form.Conjugates containing a functional cyanovirin coupled to a desiredeffector component, such as a toxin, immunological reagent, or otherfunctional reagent, can be designed even more specifically to exploitthe unique gp120-targeting properties of a cyanovirin, in accord withthe following observations.

Example 6 reveals novel gp120-directed effects of a cyanovirin.Additional insights can be gained from solid-phase ELISA experiments(Boyd et al., 1996, unpublished). For example, both C-terminalgp120-epitope-specific capture or CD4-receptor capture of gp120, whendetected either with polyclonal HIV-1-Ig or with mouse MAb to theimmunodominant, third hypervariable (V3) epitope (Matsushita et al., J.Virol. 62, 2107-2114, 1988), can be shown to be strikingly inhibited bycyanovirin-N. Generally, engagement of the CD4 receptor does notinterfere with antibody recognition of the V3 epitope, and vice versa(Moore et al., AIDS Res. Hum. Retrovir. 4, 369-379, 1988; Matsushita etal., 1988, supra). However, cyanovirin-N apparently is capable of moreglobal conformational effects on gp120, as can be demonstrated by lossof immunoreactivity at multiple, distinct, non-overlapping epitopes.

The range of antiviral activity (Boyd et al., 1996, spra) ofcyanovirin-N against diverse CD4⁺-tropic immunodeficiency virus strainsin various target cells is remarkable; diverse strains of HIV-1, HIV-2,and SIV can be shown to be similarly sensitive to cyanovirin; clinicalisolates and laboratory strains typically will show essentiallyequivalent sensitivity (for further illustration, see Example 7).Cocultivation of chronically infected and uninfected CEM-SS cells withcyanovirin-N will show that the protein will not inhibit viralreplication, but will cause a concentration-dependent inhibition ofcell-to-cell fusion and virus transmission; similar results from bindingand fusion inhibition assays employing HeLa-CD4-LTR-β-galactosidasecells can be shown consistent with cyanovirin-N inhibition of virus-celland/or cell-cell binding (Boyd, et al., 1996, supra). Example 8.illustrates the construction of a conjugate DNA coding sequence andexpression thereof to provide a cyanovirin-toxin conjugate thatselectively targets and kills HIV-infected cells.

The antiviral, e.g., anti-HIV, activity of the cyanovirins andconjugates thereof can be further demonstrated in a series ofinterrelated in vitro antiviral assays (Gulakowski et al., J. Virol.Methods 33, 87-100, 1991), which reasonably predict antiviral activityin humans. These assays measure the ability of compounds to prevent thereplication of HIV and/or the cytopathic effects of IIIV on human targetcells. These measurements directly correlate with the pathogenesis ofHIV-induced disease in vivo. The results of the analysis of theantiviral activity of cyanovirins or conjugates, as set forth in Example5 and as illustrated in FIGS. 8, 9 and 10, predict antiviral activity ofthese products in vivo in humans and, therefore, further establish theutility of the cyanovirins and conjugates thereof. Also, ex vivo methodsof use of cyanovirins and conjugates (e.g., see results set forth inExample 5, and in FIGS. 6 and 7), provide even broader utility.

The cyanovirins and conjugates thereof can be shown to inhibit a virus,specifically a retrovirus, such as the human immunodeficiency virus,i.e., HIV-1 or HIV-2. The cyanovirins and conjugates of the presentinvention can be used to inhibit other retroviruses as well as otherviruses. Examples of viruses that can be treated include, but are notlimited to, Type C and Type D retroviruses, HTLV 1, HTLV-2, HIV, FLV,SIV, MLV, BLV, BIV, equine infectious virus, anemia virus, avian sarcomaviruses, such as Rous sarcoma virus (RSV), hepatitis type A, B, non-Aand non-B viruses, arboviruses, varicella viruses, measles, mumps andrubella viruses.

Cyanovirins and conjugates thereof comprise proteins and, as such, areparticularly susceptible to hydrolysis of amide bonds (e.g., catalyzedby peptidases) and disruption of essential disulfide bonds or formationof inactivating or unwanted disulfide linkages (Carone et al., J. Lab.Clin. Med. 100, 1-14, 1982). There are various ways to alter molecularstructure, if necessary, to provide enhanced stability to the cyanovirinor conjugate thereof (Wunsch, Biopolymers 22, 493-505, 1983; Samanen, inPolymeric Materials in Medication, Gebelein et al., eds., Plenum Press:New York, 1985, pp. 227-242), which, in some circumstances, may beessential for preparation and use of pharmaceutical compositionscontaining cyanovirins or conjugates thereof for therapeutic orprophylactic applications against viruses, e.g., HIV. Possible optionsfor useful chemical modifications of a cyanovirin or conjugate thereofinclude, but are not limited to, the following (adapted from Samanen, J.M., 1985, supra): (a) olefin substitution, (b) carbonyl reduction, (c)D-amino acid substitution, (d) N α-methyl substitution, (e) C α-methylsubstitution, (f) C α-C′-methylene insertion, (g) dehydro amino acidinsertion, (h) retro-inverso modification, (i) N-terminal to C-terminalcyclization, and (j) thiomethylene modification. Cyanovirins andconjugates thereof also can be modified by covalent attachment ofcarbohydrate and polyoxyethylene derivatives, which are expected toenhance stability and resistance to proteolysis (Abuchowski et al., inEnzymes as Drugs, Holcenberg et al., eds., John Wiley: New York, 1981,pp. 367-378).

Other important general considerations for design of delivery systemsand compositions, and for routes of administration, for protein drugs,such as cyanovirins and conjugates thereof (Eppstein, CRC Crit. Rev.Therapeutic Drug Carrier Systems 5, 99-139, 1988; Siddiqui et al., CRCCrit. Rev. Therapeutic Druo Carrier Svstems 3, 195-208, 1987); Banga etal., Int. J. Pharmaceutics 48, 15-50, 1988; Sanders, Eur. J. Drug Metab.Pharmacokinetics 15, 95-102, 1990; Verhoef, Eur. J. Drug Metab.Pharmacokinetics 15, 83-93, 1990), also apply. The appropriate deliverysystem for a given cyanovirin or conjugate thereof will depend upon itsparticular nature, the particular clinical application, and the site ofdrug action. As with any protein drug, oral delivery of a cyanovirin ora conjugate thereof will likely present special problems, due primarilyto instability in the gastrointestinal tract and poor absorption andbioavailability of intact, bioactive drug therefrom. Therefore,especially in the case of oral delivery, but also possibly inconjunction with other routes of delivery, it will be necessary to usean absorption-enhancing agent in combination with a given cyanovirin orconjugate thereof. A wide variety of absorption-enhancing agents havebeen investigated and/or applied in combination with protein drugs fororal delivery and for delivery by other routes (Verhoef, 1990, supra;van Hoogdalem, Pharmac. Ther. 44, 407-443, 1989; Davis, J. Pharm.Pharmacol. 44(Suppl. 1), 186-190, 1992). Most commonly, typicalenhancers fall into the general categories of (a) chelators, such asEDTA, salicylates, and N-acyl derivatives of collagen, (b) surfactants,such as lauryl sulfate and polyoxyethylene-9-lauryl ether, (c) bilesalts, such as glycholate and taurocholate, and derivatives, such astaurodihydrofusidate, (d) fatty acids, such as oleic acid and capricacid, and their derivatives, such as acylcamitines, monoglycerides, anddiglycerides, (e) non-surfactants, such as unsaturated cyclic ureas, (f)saponins, (g) cyclodextrins, and (h) phospholipids.

Other approaches to enhancing oral delivery of protein drugs, such asthe cyanovirins and conjugates thereof, can include the aforementionedchemical modifications to enhance stability to gastrointestinal enzymesand/or increased lipophilicity. Alternatively, the protein drug can beadministered in combination with other drugs or substances that directlyinhibit proteases and/or other potential sources of enzymaticdegradation of proteins. Yet another alternative approach to prevent ordelay gastrointestinal absorption of protein drugs, such as cyanovirinsor conjugates, is to incorporate them into a delivery system that isdesigned to protect the protein from contact with the proteolyticenzymes in the intestinal lumen and to release the intact protein onlyupon reaching an area favorable for its absorption. A more specificexample of this strategy is the use of biodegradable microcapsules ormicrospheres, both to protect vulnerable drugs from degradation, as wellas to effect a prolonged release of active drug (Deasy, inMicroencapsulation and Related Processes, Swarbrick, ed., MarcellDekker, Inc.: New York, 1984, pp. 1-60, 88-89, 208-211). Microcapsulesalso can provide a useful way to effect a prolonged delivery of aprotein drug, such as a cyanovirin or conjugate thereof, after injection(Maulding, J. Controlled Release 6, 167-176, 1987).

Given the aforementioned potential complexities of successful oraldelivery of a protein drug, it is preferred in many situations that thecyanovirins and conjugates thereof be delivered by one of the numerousother potential routes of delivery of a protein drug. These routesinclude intravenous, intraarterial, intrathecal, intracisternal, buccal,rectal, nasal, pulmonary, transdermal, vaginal, ocular, and the like(Eppstein, 1988, supra; Siddiqui et al., 1987, supra; Banga et al.,1988, supra; Sanders, 1990, supra; Verhoef, 1990, supra; Barry, inDelivery Systems for Peptide Drugs, Davis et al., eds., Plenum Press:New York, 1986, pp. 265-275; Patton et al., Adv. Drug Delivery Rev. 8,179-196, 1992). With any of these routes, or, indeed, with any otherroute of administration or application, a protein drug, such as acyanovirin or conjugate thereof, may initiate an immunogenic reaction.In such situations it may be necessary to modify the molecule in orderto mask immunogenic groups. It also can be possible to protect againstundesired immune responses by judicious choice of method of formulationand/or administration. For example, site-specific delivery can beemployed, as well as masking of recognition sites from the irnmunesystem by use or attachment of a so-called tolerogen, such aspolyethylene glycol, dextran, albumin, and the like (Abuchowski et al.,1981, supra; Abuchowski et al., J. Biol. Chem. 252, 3578-3581, 1977;Lisi et al., J. Appl. Biochem. 4, 19-33, 1982; Wileman et al., J. Pharm.Pharmacol. 38, 264-271, 1986). Such modifications also can haveadvantageous effects on stability and half-life both in vivo and exvivo. Other strategies to avoid untoward immune reactions also caninclude the induction of tolerance by administration initially of onlylow doses. In any event, it will be apparent from the present disclosureto one skilled in the art that for any particular desired medicalapplication or use of a cyanovirin or conjugate thereof, the skilledartisan can select from any of a wide variety of possible compositions,routes of administration, or sites of application, whatever isadvantageous.

Accordingly, the antiviral cyanovirins and conjugates thereof can beformulated into various compositions for use either in therapeutictreatment methods for virally, e.g., HIV, infected individuals, or inprophylactic methods against viral, e.g., HIV, infection of uninfectedindividuals.

Thus, a composition comprising a cyanovirin or cyanovirin conjugate,especially a pharmaceutical composition comprising an antiviraleffective amount of an isolated and purified cyanovirin or cyanovirinconjugate and a pharmaceutically acceptable carrier is provided. Insteadof, or in addition to, the aforementioned isolated and purifiedcyanovirin or cyanovirin conjugate, the composition can comprise viablehost cells transformed to directly express a cyanovirin or conjugatethereof in vivo. The composition further can comprise an antiviraleffective amount of at least one additional antiviral compound otherthan a cyanovirin or conjugate thereof. Suitable antiviral compoundsinclude AZT, ddI, ddC, gancyclovir, fluorinated dideoxynucleosides,nevirapine, R82913, Ro 31-8959, BI-RJ-70, acyclovir, α-interferon,recombinant sCD4, michellamines, calanolides, nonoxynol-9, gossypol andderivatives thereof, and gramicidin. The cyanovirin used in thepharmaceutical composition can be isolated and purified from naturallyoccurring organisms or from genetically engineered organisms. Similarly,cyanovirin conjugates can be derived from genetically engineeredorganisms or from chemical coupling

The compositions can be used to treat a virally infected animal, such asa human. The compositions are particularly useful for inhibiting thegrowth or replication of a virus, such as a retrovirus, in particular ahuman immunodeficiency virus, specifically HIV-1 and HIV-2. Thecompositions are useful in the therapeutic or prophylactic treatment ofanimals, such as humans, who are infected with a virus or who are atrisk for viral infection, respectively. The compositions also can beused to treat objects or materials, such as medical equipment, supplies,or fluids, including biological fluids, such as blood, blood products,and tissues, to prevent viral infection of an animal, such as a human.Such compositions also are useful to prevent sexual transmission ofviral infections, e.g., HIV, which is the primary way in which theworld's AIDS cases are contracted (Merson, 1993, supra).

Potential virucides used or being considered for application againstsexual transmission of HIV are very limited; present agents in thiscategory include, for example, nonoxynol-9 (Bird, AIDS 5, 791-796,1991), gossypol and derivatives (Polsky et al., Contraception 39,579-587, 1989; Lin, Antimicrob. Agents Chemother. 33, 2149-2151, 1989;Royer, Pharmacol. Res. 24, 407-412, 1991), and gramicidin (Bourinbair,Life Sci./Pharmacol. Lett. 54, PL5-9, 1994; Bourinbair et al.Contraception 49, 131-137, 1994).

In a novel approach to anti-HIV prophylaxis currently being initiatedunder the auspices of the U.S. National Institute of Allergy andInfectious Diseases (NIAID) (e.g., as conveyed by Painter, USA Today,Feb. 13, 1996), the vaginal suppository instillation of live cultures oflactobacilli is being evaluated in a 900-woman study. This study isbased especially upon observations of anti-HIV effects of certainH₂O₂-producing lactobacilli in vitro (e.g., see published abstract byHilier, from NIAID-sponsored Conference on “Advances in AIDS VaccineDevelopment”, Bethesda, MD, February 11-15, 1996). Lactobacilli readilypopulate the vagina, and indeed are a predominant bacterial populationin most healthy women (Redondo-Lopez et al., Rev. Infect. Dis. 12,856-872, 1990; Reid et al., Clin. Microbiol. Rev. 3, 335-344, 1990;Bruce and Reid, Can. J. Microbiol. 34, 339-343, 1988; Reu et al., J.Infect. Dis. 171, 1237-1243, 1995; Hilier et al., Clin. Infect. Dis.16(Suppl 4), S273-S281; Agnew et al., Sex. Transm. Dis. 22, 269-273,1995). Lactobacilli are also prominent, nonpathogenic inhabitants ofother body cavities such as the mouth, nasopharynx, upper and lowergastrointestinal tracts, and rectum.

It is well-established that lactobacilli can be readily transformedusing available genetic engineering techniques to incorporate a desiredforeign DNA coding sequence, and that such lactobacilli can be made toexpress a corresponding desired foreign protein (see, e.g., Hols et al.,Appl. and Environ. Microbiol. 60, 1401-1413, 1994). Therefore, withinthe context of the present disclosure, it will be appreciated by oneskilled in the art that viable host cells containing a DNA sequence orvector as described above , and expressing an above-described protein,can be used directly as the delivery vehicle for a cyanovirin orconjugate thereof to the desired site(s) in vivo. Preferred host cellsfor such delivery of cyanovirins or conjugates thereof directly todesired site(s), such as, for example, to a selected body cavity, cancomprise bacteria. More specifically, such host cells can comprisesuitably engineered strain(s) of lactobacilli, enterococci, or othercommon bacteria, such as E. coli, normal strains of which are known tocommonly populate body cavities. More specifically yet, such host cellscan comprise one or more selected nonpathogenic strains of lactobacilli,such as those described by Andreu et al. (1995, supra), especially thosehaving high adherence properties to epithelial cells, such as, forexample, adherence to vaginal epithelial cells, and suitably transformedusing the above-described DNA sequences.

As reviewed by McGroarty (FEMS Immunol. Med. Microbiol. 6, 251-264,1993) the “probiotic” or direct therapeutic application of livebacteria, particularly bacteria that occur normally in nature, moreparticularly lactobacilli, for treatment or prophylaxis againstpathogenic bacterial or yeast infections of the urogenital tract, inparticular the female urogenital tract, is a well-established concept.Recently, the use of a conventional probiotic strategy, in particularthe use of live lactobacilli, to inhibit sexual transmission of HIV hasbeen suggested, based specifically upon the normal, endogenousproduction of virucidal levels of H₂O₂ and/or lactic acid and/or otherpotentially virucidal substances by certain normal strains oflactobacilli (e.g., Hilier, 1996, supra). However, the present inventiveuse of non-mammalian cells, particularly bacteria, more particularlylactobacilli, specifically engineered with a foreign gene, morespecifically a cyanovirin gene, to express an antiviral substance, morespecifically a protein, and even more specifically a cyanovirin, isheretofore unprecedented as a method of treatment of an animal,specifically a human, to prevent infection by a virus, specifically aretrovirus, more specifically HIV-1 or HIV-2.

Elmer et al. (JAMA 275, 870-876, 1996) have recently speculated that“genetic engineering offers the possibility of using microbes to deliverspecific actions or products to the colon or other mucosal surfaces . .. other fertile areas for future study include defining the mechanismsof action of various biotherapeutic agents with the possibility ofapplying genetic engineering to enhance activities.” Elmer et al. (1996,supra) further point out that the terms “probiotic” and “biotherapeuticagent” have been used in the literature to describe microorganisms thathave antagonistic activity toward pathogens in vivo; those authors morespecifically prefer the term “biotherapeutic agent” to denote“microorganisms having specific therapeutic properties.

In view of the present disclosure, one skilled in the art willappreciate that taught herein is an entirely novel type of “probiotic”or “biotherapeutic” treatment using specifically engineered strains ofmicroorganisms that do not occur in nature. Nonetheless, availableteachings concerning selection of optimal microbial strains, inparticular bacterial strains, for conventional probiotic orbiotherapeutic applications can be employed in the context of thepresent invention. For example, selection of optimal lactobacillusstrains for genetic engineering, transformation, direct expression ofcyanovirins or conjugates thereof, and direct probiotic orbiotherapeutic applications, to treat or prevent HIV infection, can bebased upon the same or similar criteria, such as those described byElmer et al. (1996, supra), typically used to select normal, endogenousor “nonengineered” bacterial strains for conventional probiotic orbiotherapeutic therapy. Furthermore, the recommendations andcharacteristics taught by McGroarty, particularly for selection ofoptimal lactobacillus strains for conventional probiotic use againstfemale urogenital infections, are pertinent to the present invention: “.. . lactobacilli chosen for incorporation into probiotic preparationsshould be easy and, if possible, inexpensive to cultivate . . . strainsshould be stable, retain viability following freeze-drying and, ofcourse, be non-pathogenic to the host . . . it is essential thatlactobacilli chosen for use in probiotic preparations should adhere wellto the vaginal epithelium . . . ideally, artificially implantedlactobacilli should adhere to the vaginal epithelium, integrate with theindigenous microorganisms present, and proliferate” (McGroarty, 1993supra). While McGroarty's teachings specifically address selections of“normal” lactobacillus strains for probiotic uses against pathogenicbacterial or yeast infections of the female urogenital tract, similarconsiderations will apply to the selection of optimal bacterial strainsfor genetic engineering and “probiotic” or “biotherapeutic” applicationagainst viral infections as particularly encompassed herein.

Accordingly, the method for the prevention of sexual transmission ofviral infection, e.g., HIV infection, comprises vaginal, rectal, oral,penile, or other topical, insertional, or instillational treatment withan antiviral effective amount of a cyanovirin and/or cyanovirinconjugate, and/or viable host cells transformed to express a cyanovirinor conjugate thereof, alone or in combination with another antiviralcompound (e.g., as described above). The compositions herein for use inthe prophylactic or therapeutic treatment methods described herein cancomprise one or more cyanovirin(s), conjugate(s) thereof, or hostcell(s) transformed to express a cyanovirin or conjugate thereof, and apharmaceutically acceptable carrier. Pharmaceutically acceptablecarriers are well-known to those skilled in the art, as are suitablemethods of administration. The choice of carrier will be determined inpart by the particular cyanovirin, or conjugate thereof, or hostcell(s), as well as by the particular method used to administer thecomposition.

One skilled in the art will appreciate that various routes ofadministering a drug are available, and, although more than one routemay be used to administer a particular drug, a particular route mayprovide a more immediate and more effective response than by anotherroute. Furthermore, one skilled in the art will appreciate that theparticular pharmaceutical carrier employed will depend, in part, uponthe particular cyanovirin, conjugate thereof, or host cell employed, andthe chosen route of administration. Accordingly, there is a wide varietyof suitable formulations of the provided composition. Formulationssuitable for oral, rectal, or vaginal administration can consist of, forexample, (a) liquid solutions or suspensions, such as an effectiveamount of the pure compound(s), and/or host cell(s) engineered toproduce directly a cyanovirin or conjugate thereof, dissolved orsuspended in diluents, such as water, culture medium, or saline, (b)capsules, suppositories, sachets, tablets, lozenges, or pastilles, eachcontaining a predetermined amount of the active ingredient(s), assolids, granules, or freeze-dried cells, and (c) oil-in-water emulsionsor water-in-oil emulsions. Tablet forms can include one or more oflactose, mannitol, corn starch, potato starch, microcrystallinecellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellosesodium, talc, magnesium stearate, stearic acid, and other excipients,colorants, diluents, buffering agents, moistening agents, preservatives,flavoring agents, and pharmacologically compatible carriers. Lozengescan comprise the active ingredient in a flavor, for example sucrose andacacia or tragacanth, while pastilles can comprise the active ingredientin an inert base, such as gelatin and glycerin, or sucrose and acacia.Suitable formulations for oral or rectal delivery also can beincorporated into synthetic and natural polymeric microspheres, or othermeans to protect the agents of the present invention from degradationwithin the gastrointestinal tract (see, for example, Wallace et al.,Science 260, 912-915, 1993).

Formulations for rectal or vaginal administration can be presented as asuppository with a suitable aqueous or nonaqueous base; the latter cancomprise, for example, cocoa butter or a salicylate. Furthermore,formulations suitable for vaginal administration can be presented aspessaries, suppositories, tampons, creams, gels, pastes, foams, or sprayformulas containing, in addition to the active ingredient, such as, forexample, freeze-dried lactobacilli genetically engineered to directlyproduce a cyanovirin or conjugate thereof of the present invention, suchcarriers as are known in the art to be appropriate. Similarly, theactive ingredient can be combined with a lubricant as a coating on acondom.

The cyanovirins, conjugates thereof, or host cells expressingcyanovirins or conjugates thereof, alone or in combination with otherantiviral compounds, can be made into aerosol formulations to beadministered via inhalation. These aerosol formulations can be placedinto pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen and the like.

The cyanovirins or conjugates thereof, alone or in combinations withother antiviral compounds or absorption modulators, can be made intosuitable formulations for dermal application and absorption (Wallace etal., 1993, supra). Transdermal electroporation or iontophoresis also canbe used to promote and/or control the systemic delivery of theabove-described compounds and/or compositions through the skin (see,e.g., Theiss et al., Meth. Find. Exp. Clin. Pharmacol. 13, 353-359,1991).

Formulations suitable for topical administration include creams,emulsions, gels, and the like containing, in addition to the activeingredient, such carriers as are known in the art, as well asmouthwashes comprising the active ingredient in a suitable liquidcarrier.

Formulations suitable for parenteral administration include aqueous andnonaqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and nonaqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example, water, for injections, immediatelyprior to use. Extemporaneous injection solutions and suspensions can beprepared from sterile powders, granules, and tablets of the kindpreviously described.

Formulations comprising a cyanovirin or cyanovirin conjugate suitablefor virucidal (e.g., against HIV) sterilization of inanimate objects,such as medical supplies or equipment, laboratory equipment andsupplies, instruments, devices, and the like, can be, for example,selected or adapted as appropriate, by one skilled in the art, from anyof the aforementioned compositions or formulations. The cyanovirin orconjugate thereof can be produced by recombinant DNA technology or bychemical coupling of a cyanovirin with an effector molecule as describedabove. Preferably, the cyanovirin, or conjugate thereof, is produced byrecombinant DNA technology. Similarly, formulations of cyanovirinsand/or conjugates thereof, suitable for ex vivo virucidal sterilizationof blood, blood products, sperm, or other bodily products or tissues, orany other solution, suspension, emulsion, or any other material whichcan be administered to a patient in a medical procedure, can be selectedor adapted as appropriate by one skilled in the art, from any of theaforementioned compositions or formulations. However, suitableformulations for such ex vivo applications or for virucidal treatment ofinanimate objects are by no means limited to any of the aforementionedformulations or compositions. One skilled in the art will appreciatethat a suitable or appropriate formulation can be selected, adapted, ordeveloped based upon the particular application at hand.

For ex vivo uses, such as virucidal treatments of inanimate objects ormaterials, blood or blood products, or tissues, the amount ofcyanovirin, or conjugate or composition thereof, to be employed shouldbe sufficient that any virus or virus-producing cells present will berendered noninfectious or will be destroyed. For example, for HIV, thiswould require that the virus and/or the virus-producing cells be exposedto concentrations of cyanovirin-N in the range of 0.1-1000 nM. Similarconsiderations apply to in vivo applications. Therefore, the phrase“antiviral effective amount” or “virucidal effective amount” is usedgenerally to describe the amount of a particular cyanovirin, conjugatethereof, or composition thereof required for antiviral efficacy in anygiven application.

For in vivo uses, the dose of a cyanovirin, conjugate thereof, hostcells producing a cyanovirin or conjugate thereof, or compositionthereof, administered to an animal, particularly a human, in the contextof the present invention should be sufficient to effect a prophylacticand/or therapeutic response in the individual over a reasonabletime-frame. The dose used to achieve a desired virucidal concentrationin vivo (e.g., 0.1-1000 nM) will be determined by the potency of theparticular cyanovirin or conjugate thereof, or of the cyanovirin and/orconjugate production of the host cells employed, the severity of thedisease state of infected individuals, as well as, in the case ofsystemic administration, the body weight and age of the infectedindividual. The effective or virucidal dose also will be determined bythe existence of any adverse side-effects that may accompany theadministration of the particular cyanovirin, conjugate thereof, hostcells producing a cyanovirin or conjugate thereof, or compositionthereof, employed. It is always desirable, whenever possible, to keepadverse side effects to a minimum.

The dosage can be in unit dosage form, such as a tablet or capsule. Theterm unit dosage form” as used herein refers to physically discreteunits suitable as unitary dosages for human and animal subjects, eachunit containing a predetermined quantity of a cyanovirin, conjugatethereof, or amount of host cells producing a cyanovirin or conjugatethereof, alone or in combination with other antiviral agents, calculatedin a quantity sufficient to produce the desired effect in associationwith a pharmaceutically acceptable carrier, diluent, or vehicle.

The specifications for the unit dosage forms of the present inventiondepend on the particular cyanovirin, conjugate, host cells, orcomposition thereof employed, and the effect to be achieved, as well asthe pharmacodynamics associated with each cyanovirin, conjugate, hostcells, or composition thereof in the treated animal. The doseadministered should be an “antiviral effective amount” or “virucidaleffective amount” or an amount necessary to achieve an “effectivevirucidal level” in the individual animal, e.g., the human patient.

Since the “effective virucidal level” is used as the preferred endpointfor dosing, the actual dose and schedule can vary, depending uponinterindividual differences in pharmacokinetics, drug distribution, andmetabolism. The “effective virucidal level” can be defined, for example,as the blood or tissue level (e.g., 0.1-1000 nM) desired in the patientthat corresponds to a concentration of one or more cyanovirins orconjugates thereof, which inhibits a virus, such as HIV-1 and/or HIV-2,in an assay known to predict for clinical antiviral activity of chemicalcompounds and biological agents. The “effective virucidal level” foragents of the present invention also can vary when the cyanovirin,conjugate, or composition thereof, is used in combination with AZT orother known antiviral compounds or combinations thereof.

One skilled in the art can easily determine the appropriate dose,schedule, and method of administration for the exact formulation of thecomposition being used, in order to achieve the desired “effectivevirucidal level” in the individual patient. One skilled in the art alsocan readily determine and use an appropriate indicator of the “effectorconcentration” of the compounds of the present invention by a direct(e.g., analytical chemical analysis) or indirect (e.g., with surrogateindicators such as p24 or RT) analysis of appropriate patient samples(e.g., blood and/or tissues).

In the treatment of some virally infected individuals, it may bedesirable to utilize a “mega-dosing” regimen, wherein a large dose of aselected cyanovirin or conjugate thereof is administered, and timethereafter is allowed for the drug to act, and then a suitable reagentis administered to the individual to inactivate the drug.

The pharmaceutical composition can contain other pharmaceuticals, inconjunction with the cyanovirin, conjugate thereof, or host cellsproducing a cyanovirin or conjugate thereof, when used totherapeutically treat a viral infection, such as that which causes AIDS.Representative examples of these additional pharmaceuticals includeantiviral compounds, virucides, immunomodulators, immunostimulants,antibiotics, and absorption enhancers. Exemplary antiviral compoundsinclude AZT, ddI, ddC, gancylclovir, fluorinated dideoxynucleosides,nonnucleoside analog compounds, such as nevirapine (Shih et al., PNAS88, 9878-9882, 1991), TIBO derivatives, such as R82913 (White et al.,Antiviral Res. 16, 257-266,1991), BI-RJ-70 (Merigan, Am. J. Med. 90(Suppl.4A), 8S-17S, 1991), michellamines (Boyd et al., J. Med. Chem. 37,1740-1745, 1994), and calanolides (Kashman et al., J. Med. Chem. 35,2735-2743, 1992), nonoxynol-9, gossypol and derivatives, and gramicidin(Bourinbair et al., 1994, supra). Exemplary immunomodulators andimmunostimulants include various interleukins, sCD4, cytokines, antibodypreparations, blood transfusions, and cell transfusions. Exemplaryantibiotics include antifungal agents, antibacterial agents, andanti-Pneumocystitis carnii agents. Exemplary absorption enhancersinclude bile salts and other surfactants, saponins, cyclodextrins, andphospholipids (Davis, 1992, supra).

The administration of a cyanovirin or conjugate thereof with otherantiretroviral agents and particularly with known RT inhibitors, such asddC, AZT, ddI, ddA, or other inhibitors that act against other HIVproteins, such as anti-TAT agents, is expected to inhibit most or allreplicative stages of the viral life cycle. The dosages of ddC and AZTused in AIDS or ARC patients have been published. A virustatic range ofddC is generally between 0.05 μM to 1.0 μM. A range of about 0.005-0.25mg/kg body weight is virustatic in most patients. The preliminary doseranges for oral administration are somewhat broader, for example 0.001to 0.25 mg/kg given in one or more doses at intervals of 2, 4, 6, 8, 12,etc. hours. Currently, 0.01 mg/kg body weight ddC given every 8 hrs, ispreferred. When given in combined therapy, the other antiviral compound,for example, can be given at the same time as the cyanovirin, orconjugate thereof, or the dosing can be staggered as desired. Thedifferent drugs also can be combined in a composition. Doses of each canbe less when used in combination than when either is used alone.

It also will be appreciated by one skilled in the art that a DNAsequence of a cyanovirin or conjugate thereof can be inserted ex vivointo mammalian cells previously removed from a given animal, inparticular a human. Such transformed autologous or homologous hostcells, reintroduced into the animal or human, will express directly thecorresponding cyanovirin or conjugate in vivo. The feasibility of such atherapeutic strategy to deliver a therapeutic amount of an agent inclose proximity to the desired target cells and pathogens (e.g., to thevirus, more particularly to the retrovirus, specifically to HIV and itsenvelope glycoprotein gp120), has been demonstrated in studies withcells engineered ex vivo to express sCD4 (Morgan et al., 1994, supra).As an alternative to ex vivo insertion of the DNA sequences of thepresent invention, such sequences can be inserted into cells directly invivo, such as by use of an appropriate viral or other suitable vector.Such cells transfected in vivo may be expected to produce antiviralamounts of cyanovirin or conjugate thereof directly in vivo. Example 9illustrates the transformation and expression of a cyanovirin by amammalian cell.

Given the present disclosure, it will be additionally appreciated that aDNA sequence corresponding to a cyanovirin or conjugate thereof can beinserted into suitable nonmammalian host cells, and that such host cellswill express therapeutic or prophylactic amounts of a cyanovirin orconjugate thereof directly in vivo within a desired body compartment ofan animal, in particular a human. Example 3 illustrates thetransformation and expression of effective virucidal amounts of acyanovirin in a non-mammalian cell, more specifically a bacterial cell.Example 10 illustrates the transformation and expression of a cyanovirinin a non-mammalian cell, specifically a yeast cell.

In a preferred embodiment of the present invention, a method offemale-controllable prophylaxis against HIV infection comprises theintravaginal administration and/or establishment of, in a female human,a persistent intravaginal population of lactobacilli that have beentransformed with a coding sequence of the present invention to produce,over a prolonged time, effective virucidal levels of a cyanovirin orconjugate thereof, directly on or within the vaginal and/or cervicaland/or uterine mucosa. It is noteworthy that both the World HealthOrganization (WHO), as well as the U.S. National Institute of Allergyand Infectious Diseases, have pointed to the need for development offemale-controlled topical microbicides, suitable for blocking thetransmission of HIV, as an urgent global priority (Lange et al., Lancet341, 1356, 1993; Fauci, NIAID News, Apr. 27, 1995).

Also provided are antibodies directed to the above-described proteins.More specifically provided by the present invention is ananti-cyanovirin antibody, which has an internal image of gp120 of aprimate immunodeficiency virus. Preferably, the antibody can competewith gp120 of a primate immunodeficiency virus for binding to acyanovirin. In this regard, the primate immunodeficiency viruspreferably is HIV-1 or HIV-2 and the cyanovirin preferably comprises SEQID NO: 2.

The availability of antibodies to any given protein is highlyadvantageous, as it provides the basis for a wide variety of qualitativeand quantitative analytical methods, separation and purificationmethods, and other useful applications directed to the subject proteins.Antibodies, in particular antibodies specifically binding to a protein,i.e., a cyanovirin, such as that of SEQ ID NO: 2, as described above,can be prepared using well-established methodologies (e.g., such as themethodologies described in detail by Harlow and Lane, in Antibodies. ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,1988, pp. 1-725). Such antibodies can comprise both polyclonal andmonoclonal antibodies. Anti-idiotypic antibodies also can be generatedin accordance with methods known in the art (see, for example, Benjamin,In Immunology: a short course, Wiley-Liss, N.Y., 1996, pp. 436-437;Kuby, In Immunology, 3rd. ed., Freeman, N.Y., 1997, pp. 455-456;Greenspan et al., FASEB J. 7, 437-443, 1993; and Poskitt, Vaccine, 9,792-796, 1991).

Such antibodies can be obtained and employed either in solution-phase orcoupled to a desired solid-phase matrix. Having in hand such antibodies,one skilled in the art will further appreciate that such antibodies,using well-established procedures (e.g., such as described by Harlow andLane (1988, supra), are useful in the detection, quantification, orpurification of a cyanovirin, a conjugate thereof, or a host celltransformed to produce a cyanovirin or conjugate thereof. Example 11further illustrates an antibody that specifically binds to a cyanovirin.Such antibodies are also useful in a method of prevention and treatmentof a viral infection in an animal as provided herein.

In view of the above, a cyanovirin can be administered to an animal. Theanimal generates anti-cyanovirin antibodies. Among the anti-cyanovirinantibodies generated or induced in the animal are antibodies that havean internal image of gp120. In accordance with well-known methods,polyclonal or monoclonal antibodies can be obtained, isolated andselected. Selection of an anti-cyanovirin antibody that has an internalimage of gp120 can be based upon competition between the anti-cyanovirinantibody and gp120 for binding to a cyanovirin, or upon the ability ofthe anti-cyanovirin antibody to bind to a free cyanovirin as opposed toa cyanovirin bound to gp120. Such an anti-cyanovirin antibody can beadministered to an animal to prevent or treat a viral infection inaccordance with methods provided herein. Although nonhumananti-idiotypic antibodies, such as an anti-cyanovirin antibody that hasan internal image of gp120 and, therefore, is anti-idiotypic to gp120,are proving useful as vaccine antigens in humans, their favorableproperties might, in certain instances, be further enhanced and/or theiradverse properties further diminished, through “humanization”strategies, such as those recently reviewed by Vaughan, Nature Biotech.,16, 535-539, 1998. Alternatively, a cyanovirin can be directlyadministered to an animal for prevention or treatment of a viralinfection in accordance with methods provided herein such that thetreated animal, itself, generates an anti-cyanovirin antibody that hasan internal image of gp120. The production of anti-idiotypic antibodies,such as an anti-cyanovirin antibody that has an internal image of gp120and, therefore, is anti-idiotypic to gp120, in an animal to be treatedis known as “anti-idiotype induction therapy,” and is described byMadiyalakan et al., Hybridoma, 14, 199-203, 1995, for example.

In view of the above, the present invention provides a method ofpreventing or treating a viral infection in an animal. The methodcomprises administering to the animal an anti-cyanovirin antibody in anamount sufficient to induce in the animal an immune response to a virussufficient to prevent or treat an infection of the animal with thevirus. The anti-cyanovirin antibody has an internal image of gp120 of aprimate immunodeficiency virus. Preferably, the antibody can competewith gp120 of a primate immunodeficiency virus for binding to acyanovirin. In this regard, the primate immunodeficiency viruspreferably is HIV-1 or HIV-2 and the cyanovirin preferably comprises SEQID NO: 2.

Also provided by the present invention is another method of preventingor treating a viral infection in an animal. The method comprisesadministering to the animal a cyanovirin, which binds gp120 of a primateimmunodeficiency virus, in an amount sufficient to induce in the animalan anti-cyanovirin antibody in an amount sufficient to induce an immuneresponse to a virus sufficient to treat or prevent an infection of theanimal with the virus. Preferably, the anti-cyanovirin antibody cancompete with gp120 of a primate immunodeficiency virus for binding to acyanovirin. In this regard, the primate immunodeficiency viruspreferably is HIV-1 or HIV-2 and the cyanovirin preferably comprises SEQID NO: 2.

With respect to the above methods, sufficient amounts can be determinedin accordance with methods known in the art. Similarly, the sufficiencyof an immune response in the prevention or treatment of a viralinfection in an animal also can be assessed in accordance with methodsknown in the art.

Either one of the above methods can further comprise concurrent, pre- orposttreatment with an adjuvant to enhance the immune response (see, forexample, Harlow et al., 1988, supra).

The above-described nucleic acid sequences, cyanovirins, conjugates,host cells, antibodies, compositions, and methods are further describedin the context of the following examples. These examples serve toillustrate further the present invention and are not intended to limitthe scope of the invention.

EXAMPLES Example 1

This example details the anti-HIV bioassay-guided isolation andelucidation of pure cyanovirin from aqueous extracts of the culturedcyanobacterium, Nostoc ellipsosporum.

The method described in Weislow et al. (1989, supra) was used to monitorand direct the isolation and purification process. Cyanobacterialculture conditions, media, and classification were as describedpreviously (Patterson, J. Phycol. 27, 530-536, 1991). Briefly, thecellular mass from a unialgal strain of Nostoc ellipsosporum (cultureQ68D170) was harvested by filtration, freeze-dried, and extracted withMeOH—CH₂Cl₂ (1:1) followed by H₂O. Bioassay indicated that only the H₂Oextract contained HIV-inhibitory activity. A solution of the aqueousextract (30 mg/ml) was treated by addition of an equal volume of ethanol(EtOH). The resulting 1:I H₂O-EtOH solution was kept at −20° C. for 15hrs. Then, the solution was centrifuged to remove precipitated materials(presumably, high molecular weight biopolymers). The resultingHIV-inhibitory supernatant was evaporated, fractionated by reverse-phasevacuum-liquid chromatography (Coll et al., J. Nat. Prod. 49, 934-936,1986; Pelletier et al., J. Nat. Prod. 49, 892-900, 1986) on wide-pore C₄packing (300Å, BakerBond WP-C₄), and eluted with increasingconcentrations of methanol (MeOH) in H₂O. Anti-HIV activity wasconcentrated in the material eluted with MeOH-H₂O (2:1). SDS-PAGEanalysis of this fraction showed one main protein band, with a relativemolecular mass (Mr) of approximately 10 kDa. Final purification wasachieved by repeated reverse-phase HPLC on 1.9×15 cm μBondapak C₁₈(Waters Associates) columns eluted with a gradient of increasingconcentration of acetonitrile in H₂O. The mobile phase contained 0.05%(v/v) TFA, pH=2. Eluted proteins were detected by UV absorption at 206,280, and 294 nm with a rapid spectral detector (Pharmacia LKB model2140). Individual -fractions were collected, pooled based on the UVchromatograni, and lyophilized. Pooled HPLC fractions were subjected toSDS-PAGE under reducing conditions (Laemmli, Nature 227, 680-685, 1970),onventional amino acid analysis, and testing for anti-HIV activity.

FIG. 1A is a graph of OD (206 nm) versus time (min), which shows the μBondapak C₁₈ HPLC chromatogram of nonreduced cyanovirin eluted with alinear CH₃CN/H₂O gradient (buffered with 0.05% TFA) from 28-38% CH₃CN.FIG. 1D is a graph of OD (206 nm) versus time (min), which shows thechromatogram of cyanovirin that was first reduced with β-mercaptoethanoland then separated under identical HPLC conditions. HPLC fractions fromthe two runs were collected as indicated. 10% aliquots of each fractionwere lyophilized, made up in 100 μl 3:1 H₂O/DMSO, and assessed foranti-HIV activity in the XTT assay. FIG. 1B is a bar graph of maximumdilution for 50% protection versus HPLC fraction, which illustrates themaximum dilution of each fraction that provided 50% protection from thecytopathic effects of HIV infection for the nonreduced cyanovirin HPLCfractions. Corresponding anti-HIV results for the HPLC fractions fromreduced cyanovirin are shown in FIG. 1E, which is a bar graph of maximumdilution for 50% protection versus HPLC fraction. 20% aliquots ofselected HPLC fractions were analyzed by SDS-PAGE. The results from thenonreduced HPLC fractions are shown in FIG. IC, and those from thereduced HPLC fractions are shown in FIG. 1F.

In the initial HPLC separation, using a linear gradient from 30-50%CH₃CN, the anti-HIV activity coeluted with the principal UV-absorbingpeak at approximately 33% CH₃CN. Fractions corresponding to the activepeak were pooled and split into two aliquots.

Re-injection of the first aliquot under similar HPLC conditions, butwith a linear gradient from 28-38% CH₃CN, resolved the active materialinto two closely eluting peaks at 33.4 and 34.0% CH₃CN. The anti-HIVactivity profile of the fractions collected during this HPLC run (asshown in FIG. 1B) corresponded with the two UV peaks (as shown in FIG.1A). SDS-PAGE of fractions collected under the individual peaks showedonly a single protein band (as shown in FIG. 1C).

The second aliquot from the original HPLC separation was reduced withβ-mercaptoethanol prior to re-injection on the HPLC. Using an identical28-38% gradient, the reduced material gave one principal peak (as shownin FIG. 1D) that eluted later in the run with 36.8% CH₃CN. Only a traceof anti-HIV activity was detected in the HPLC fractions from the reducedmaterial (as shown in FIG. 1E).

The two closely eluting HPLC peaks of the nonreduced material (FIG. 1A)gave only one identical band on SDS-PAGE (run under reducing conditions)(FIG. 1C), and reduction with β-mercaptoethanol resulted in an HPLC peakwith a longer retention time than either of the nonreduced peaks (FIG.1F). This indicated that disulfides were present in the native protein.Amino acid analysis of the two active peaks showed they had virtuallyidentical compositions. It is possible that the two HPLC peaks resultedfrom cis/trans isomerism about a proline residue or frommicroheterogeneity in the protein sample that was not detected in eitherthe amino acid analysis or during sequencing. The material collected asthe two HIV-inhibitory peaks was combined for further analyses and wasgiven the name cyanovirin-N.

Example 2

This example illustrates the synthesis of cyanovirin genes.

The chemically deduced amino acid sequence of cyanovirin-N wasback-translated to obtain a DNA coding sequence. In order to facilitateinitial production and purification of recombinant cyanovirin-N, acommercial. expression vector (pFLAG-1, from InternationalBiotechnologies, Inc., New Haven, Conn.), for which reagents wereavailable for affinity purification and detection, was selected.Appropriate restriction sites for ligation to pFLAG-1, and a stop codon,were included in the DNA sequence. FIG. 2 is an example of a DNAsequence encoding a synthetic cyanovirin gene. This DNA sequence designcouples the cyanovirin-N coding region to codons for a “FLAG”octapeptide at the N-terminal end of cyanovirin, providing forproduction of a FLAG-cyanovirin fusion protein.

A flowchart for synthesis of this DNA sequence is as follows:

Design DNA Coding Sequence 327 Synthesize Oligonucleotide bp → Elements↓ Drop Dialysis Against Distilled Purification of OligosWater/Quantitation ← ↓ Dry 10 nmole of Each Oligo in Resuspend in T4 DNASpeedvac → Ligase Buffer ↓ Heat for 20 min at 65° C. to Treat with T4Polynucleotide inactivate the Kinase ← Kinase for 2 hrs at 37° C. ↓ PoolOligos and Boil for 10 min Anneal for 20 min at 70° C. → ↓ Add T4 DNALigase and Additional Cool on Ice Buffer and Incubate at 16° C. ←Overnight ↓ Phenol:Chloroform Extract/EtOH Dissolve in Distilled WaterPrecipitate → ↓ Run Low-Melting Agarose Gel Preparative PCR ← ↓ Excise327 bp Band Recover/Purify Dissolve DNA →

The DNA sequence was synthesized as 13 overlapping, complementaryoligonucleotides and assembled to form the double-stranded codingsequence Oligonucleotide elements of the synthetic DNA coding sequencewere synthesized using a dual-column nucleic acid synthesizer (Model392, Applied Biosystems Inc., Foster City, Calif.). Completedoligonucleotides were cleaved from the columns and deprotected byincubation overnight at 56° C. in concentrated ammonium hydroxide. Priorto treatment with T4 polynucleotide kinase, 33-66 mers weredrop-dialyzed against distilled water. The 13 oligonucleotidepreparations were individually purified by HPLC, and 10 nmole quantitiesof each were ligated with T4 DNA ligase into a 327 bp double-strandedDNA sequence. DNA was recovered and purified from the reaction buffer byphenol:chloroform extraction, ethanol precipitation, and further washingwith ethanol. Individual oligonucleotide preparations were pooled andboiled for 10 min to ensure denaturation. The temperature of the mixturewas then reduced to 70° C. for annealing of the complementary strands.After 20 min, the tube was cooled on ice and 2,000 units of T4 DNAligase were added together with additional ligase buffer. Ligation wasperformed overnight at 16° C. DNA was recovered and purified from theligation reaction mixture by phenol:chloroform extraction and ethanolprecipitation and washing.

The purified, double-stranded synthetic DNA was then used as a templatein a polymerase chain reaction (PCR). One μl of the DNA solutionobtained after purification of the ligation reaction mixture was used asa template. Thermal cycling was performed using a Perkin-Elmerinstrument. “Vent” thermostable DNA polymerase, restriction enzymes, T4DNA ligase, and polynucleotide kinase were obtained from New EnglandBiolabs, Beverly, MA. Vent polymerase was selected for this applicationbecause of its claimed superiority in fidelity compared to the usual Taqenzyme. The PCR reaction product was run on a 2% agarose gel in TBEbuffer. The 327 bp construct was then cut from the gel and purified byelectroelution. Because it was found to be relatively resistant todigestion with Hind III and Xho I restriction enzymes, it was initiallycloned using the pCR-Script system (Stratagene). Digestion of a plasmidpreparation from one of these clones yielded the coding sequence, whichwas then ligated into the multicloning site of the pFLAG-1 vector.

E. coli were transformed with the pFLAG-construct, and recombinantclones were identified by analysis of restriction digests of plasmidDNA. Sequence analysis of one of these selected clones indicated thatfour bases deviated from the intended coding sequence. This includeddeletion of three bases coding for one of four cysteine residuescontained in the protein and an alteration of the third base in thepreceding codon (indicated by the boxes in FIG. 2). In order to correctthese “mutations,” which presumably arose during the PCR amplificationof the synthetic template, a double-stranded “patch” was synthesized,which could be ligated into restriction sites flanking the mutations(these Bst XI and Esp1 sites are also indicated in FIG. 2). The patchwas applied and the repair was confirmed by DNA sequence analysis.

For preparation of a DNA sequence coding for native cyanovirin, theaforementioned FLAG-cyanovirin construct was subjected to site-directedmutagenesis to eliminate the codons for the FLAG octapeptide and, at thesame time, to eliminate a unique Hind III restriction site. Thisprocedure is illustrated in FIG. 3, which illustrates a site-directedmutagenesis maneuver used to eliminate codons for a FLAG octapeptide anda Hind III restriction site from the sequence of FIG. 2. A mutagenicoligonucleotide primer was synthesized, which included portions of thecodons for the Omp secretory peptide and cyanovirin, but lacked thecodons for the FLAG peptide. Annealing of this mutagenic primer, withcreation of a DNA hairpin in the template strand, and extension by DNApolymerase resulted in the generation of a new plasmid DNA lacking boththe FLAG codon sequence and the Hind III site (refer to FIG. 2 fordetails). The digestion of the plasmid DNA with Hind III resulted inlinearization of “wild-type” strands but not “mutant” strands. Sincetransformation of E. coli occurs more efficiently with circular DNA,clones could be readily selected which had the revised coding sequencewhich specified production of native cyanovirin-N directly behind theOmp secretory peptide. DNA sequencing verified the presence of theintended sequence. Site-directed mutagenesis reactions were carried outusing materials (polymerase, buffers, etc.) obtained from PharmaciaBiotech, Inc., Piscataway, N.J.

Example 3

This example illustrates the expression of synthetic cyanovirin genes.

As indicated in the following flowchart:

Recover/Purify/Dissolve PCR Product ↓ Blunt-End Clone (Stratagene Kit) ↓Plasmid Preparation on Selected Clone ↓ Digest with Hind III and Xho Iand Ligate to pFLAG-1 ↓ Transform E. coli, Isolate, and IdentifyRecombinant Clones (Experimental and BAP Control) ↓ Seed Small-ScaleShake Cultures ↓ Induce Express with IPTG ↓ Prepare Crude PeriplasmicExtract ↓ Anti-HIV Bioassay and FLAG Dot-Blot ↓

E. coli (strain DH5α) were transformed (by electroporation) with thepFLAG-1 vector containing the coding sequence for the FLAG-cyanovirin-Nfusion protein (see FIG. 2 for details of the DNA sequence). Selectedclones were seeded into small-scale shake flasks containing (LB) growthmedium with 100 μg/ml ampicillin and expanded by incubation at 37° C.Larger-scale Erlenmeyer flasks (0.5-3.0 liters) were then seeded andallowed to grow to a density of 0.5-0.7 OD₆₀₀ units. The expression ofthe FLAG-cyanovirin-N fusion protein was then induced by adding IPTG toa final concentration of 1.7 mM and continuing incubation at 30° C. for3-6 hrs. For harvesting of periplasmic proteins, bacteria were pelleted,washed, and then osmotically shocked by treatment with sucrose, followedby resuspension in distilled water. Periplasmic proteins were obtainedby sedimenting the bacteria and then filtering the aqueous supernatantthrough Whatman paper. Crude periplasmic extracts showed both anti-HIVactivity and presence of a FLAG-cyanovirin-N fusion protein by Westernor spot-blotting.

The construct for native cyanovirin-N described in Example 2 was used totransform bacteria in the same manner as described above for theFLAG-cyanovirin-N fusion protein. Cloning, expansion, induction withIPTG, and harvesting were performed similarly. Crude periplasmicextracts showed strong anti-HIV activity on bioassay.

Example 4

This example illustrates purification of recombinant cyanovirinproteins.

Using an affinity column based on an anti-FLAG monoclonal antibody(International Biotechnologies, Inc., New Haven, Conn.),FLAG-cyanovirin-N fusion protein could be purified as follows:

Periplasmic Extract of IPTG Induced Cultures ↓ Load onto Affinity Column↓ Wash Through E. coli Proteins ↓ Elute Bound Fusion Protein with EDTA ↓Dialyze Against Water and Lyophilize

The respective periplasmic extract, prepared as described in Example 3,was loaded onto 2-20 ml gravity columns containing affinity matrix andwashed extensively with PBS containing CA⁺⁺ to remove contaminatingproteins. Since the binding of the FLAG peptide to the antibody isCa⁺⁺-dependent, fusion protein could be eluted by passage of EDTAthrough the column. Column fractions and wash volumes were monitored byspot-blot analysis using the same anti-FLAG antibody. Fractionscontaining fusion protein were then pooled, dialyzed extensively againstdistilled water, and lyophilized.

For the purification of the recombinant native cyanovirin-N, thecorresponding periplasmic extract from Example 3 was subjected tostep-gradient C₄ reverse-phase, vacuum-liquid chromatography to givethree fractions: (1) eluted with 100% H₂O, (2) eluted with MeOH—H₂O(2:1), and (3) eluted with 100% MeOH. The anti-HIV activity wasconcentrated in fraction (2). Purification of the recombinantcyanovirin-N was performed by HPLC on a 1.9×15 cm μBondapak (WatersAssociates) C₁₈ column eluted with a gradient of increasingconcentration of CH₃CN in H₂O (0.05% TFA, v/v in the mobile phase). Achromatogram of the final HPLC purification on a 1×10 cm (CohensiveTechnologies, Inc.) C₄ column monitored at 280 nm is shown in FIG. 4,which is typical HPLC chromatogram during the purification of arecombinant native cyanovirin. Gradient elution, 5 ml/min, from 100% H₂Oto H₂O—CH₃CN (7:3) was carried out over 23 min with 0.05% TFA (v/v) inthe mobile phase.

Example 5

This example illustrates the anti-HIV activities of natural andrecombinant cyanovirin-N and FLAG-cyanovirin-N.

Pure proteins were initially evaluated for antiviral activity using anXTT-tetrazolium anti-HIV assay described previously (Boyd, in AIDS,Etiology, Diaonosis, Treatment and Prevention, 1988, supra; Gustafson etal., J. Med. Chem. 35, 1978-1986,1992; Weislow, 1989, supra; Gulakowski,1991, supra). The CEM-SS human lymphocytic target cell line used in allassays was maintained in RPMI 1650 medium (Gibco, Grand Island, N.Y.),without phenol red, and was supplemented with 5% fetal bovine serum, 2mM L-glutamine, and 50 μg/ml gentamicin (complete medium).

Exponentially growing cells were pelleted and resuspended at aconcentration of2.0×10⁵ cells/ml in complete medium. The Haitian variantof HIV, HTLV-III_(RF) (3.54×10⁶ SFU/ml), was used throughout. Frozenvirus stock solutions were thawed immediately before use and resuspendedin complete medium to yield 1.2×12⁵ SFU/ml. The appropriate amounts ofthe pure proteins for anti-HIV evaluations were dissolved in H₂O-DMSO(3:1), then diluted in complete medium to the desired initialconcentration. All serial drug dilutions, reagent additions, andplate-to-plate transfers were carried out with an automated Biomek 1000Workstation (Beckman Instruments, Palo Alto, Calif.).

FIGS. 5A-5C are graphs of % control versus concentration (nM), whichillustrate antiviral activities of native cyanovirin from Nostocellipsosporum (A), recombinant native (B), and recombinant FLAG-fusioncyanovirins. The graphs show the effects of a range of concentrations ofthe respective cyanovirins upon CEM-SS cells infected with HIV-1 (), asdetermined after 6 days in culture. Data points represent the percent ofthe respective uninfected, nondrug-treated control values. All threecyanovirins showed potent anti-HIV activity, with an EC₅₀ in the lownanomolar range and no significant evidence of direct cytotoxicity tothe host cells at the highest tested concentrations (up to 1.2 μM).

As an example of a further demonstration of the anti-HIV activity ofpure cyanovirin-N, a battery of interrelated anti-HIV assays wasperformed in individual wells of 96-well microtiter plates, usingmethods described in detail elsewhere (Gulakowski, 1991, supra).Briefly, the procedure was as follows. Cyanovirin solutions wereserially diluted in complete medium and added to 96-well test plates.Uninfected CEM-SS cells were plated at a density of 1×10⁴ cells in 50 μlof complete medium. Diluted HIV-1 was then added to appropriate wells ina volume of 50 μl to yield a multiplicity of infection of 0.6.Appropriate cell, virus, and drug controls were incorporated in eachexperiment. The final volume in each microtiter well was 200 μl.Quadruplicate wells were used for virus-infected cells. Plates wereincubated at 37° C. in an atmosphere containing 5% CO₂ for 4, 5, or 6days.

Subsequently, aliquots of cell-free supernatant were removed from eachwell 15 using the Biomek, and analyzed for reverse transcriptaseactivity, p24 antigen production, and synthesis of infectious virions asdescribed (Gulakowski, 1991, supra). Cellular growth or viability thenwas estimated on the remaining contents of each well using the XTT(Weislow et al., 1989, supra), BCECF (Rink et al., J. Cell Biol. 95,189-196, 1982) and DAPI (McCaffrey et al., In vitro Cell Develop. Biol.24, 247-252, 1988) assays as described (Gulakowski et al., 1991, supra).To facilitate graphical displays and comparisons of data, the individualexperimental assay results (of at least quadruplicate determinations ofeach) were averaged, and the mean values were used to calculatepercentages in reference to the appropriate controls. Standard errors ofthe mean values used in these calculations typically averaged less than10% of the respective mean values.

FIGS. 6A-6D are graphs of % control versus concentration (nM), whichillustrate anti-HIV activity of a cyanovirin in a multiparameter assayformat. Graphs 6A, 6B, and 6C show the effects of a range ofconcentrations of cyanovirin upon uninfected CEM-SS cells (◯), and uponCEM-SS cells infected with HIV-1 (), as 30 determined after 6 days inculture. Graph 6A depicts the relative numbers of viable CEM-SS cells,as assessed by the BCECF assay. Graph 6B depicts the relative DNAcontents of the respective cultures. Graph 6C depicts the relativenumbers of viable CEM-SS cells, as assessed by the XTT assay. Graph 6Dshows the effects of a range of concentrations of cyanovirin uponindices of infectious virus or viral replication as determined after 4days in culture. These indices include viral reverse transcriptase (▴),viral core protein p24 (♦), and syncytium-forming units (▪). In graphs6A, 6B, and 6C, the data are represented as the percent of theuninfected, nondrug-treated control values. In graph 6D the data arerepresented as the percent of the infected, nondrug-treated controlvalues.

As illustrated in FIG. 6, cyanovirin-N was capable of completeinhibition of the cytopathic effects of HIV-1 upon CEM-SS humanlymphoblastoid target cells in vitro; direct cytotoxicity of the proteinupon the target cells was not observed at the highest testedconcentrations. Cyanovirin-N also strikingly inhibited the production ofRT, p24, and SFU in HIV-1-infected CEM-SS cells within these sameinhibitory effective concentrations, indicating that the protein haltedviral replication.

The anti-HIV activity of the cyanovirins is extremely resilient to harshenvironmental challenges. For example, unbuffered cyanovirin-N solutionswithstood repeated freeze-thaw cycles or dissolution in organic solvents(up to 100% DMSO, MeOH, or CH₃CN) with no loss of activity. Cyanovirin-Ntolerated detergent (0.1% SDS), high salt (6 M guanidine HCl), and heattreatment (boiling, 10 min in H₂O) with no significant loss ofHIV-inhibitory activity. Reduction of the disulfides withβ-mercaptoethanol, followed immediately by C₁₈ HPLC purification,drastically reduced the cytoprotective activity of cyanovirin-N.However, solutions of reduced cyanovirin-N regained anti-HIV inhibitoryactivity during prolonged storage. When cyanovirin-N was reduced(β-mercaptoethanol, 6 M guanidine HCl, pH 8.0) but not put through C₁₈HPLC, and, instead, simply desalted, reconstituted, and assayed, itretained virtually full activity.

Example 6

This example illustrates that the HIV viral envelope gp120 is aprincipal molecular target of cyanovirin-N.

Initial experiments, employing the XTT-tetrazolium assay (Weislow etal., 1989, supra), revealed that host cells preincubated with cyanovirin(10 nM, 1 hr), then centrifuged free of cyanovirin-N, retained normalsusceptibility to HIV infection; in contrast, the infectivity ofconcentrated virus similarly pretreated, then diluted to yieldnon-inhibitory concentrations of cyanovirin-N, was essentiallyabolished. This indicated that cyanovirin-N was acting directly upon thevirus itself, i.e., acting as a direct “virucidal” agent to preventviral infectivity even before it could enter the host cells. This wasfurther confirmed in time-of-addition experiments, likewise employingthe XTT-tetrazolium assay (Weislow et al., 1989, supra), which showedthat, to afford maximum antiviral activity, cyanovirin-N had to be addedto cells before or as soon as possible after addition of virus as shownin FIG. 7, which is a graph of % uninfected control versus time ofaddition (hrs), which shows results of time-of-addition studies of acyanovirin, showing anti-HIV activity in CEM-SS cells infected withHIV-1_(RF). Introduction of cyanovirin () or ddC (▪) (10 nM and 5 μMconcentrations, respectively) was delayed by various times after initialincubation, followed by 6 days incubation, then assay of cellularviability (linegraphs) and RT (open bars, inset). Points representaverages (±S.D.) of at least triplicate determinations. In markedcontrast to the reverse transcriptase inhibitor ddC, delay of additionof cyanovirin-N by only 3 hrs resulted in little or no antiviralactivity (FIG. 7). The aforementioned results suggested thatcyanovirin-N inhibited HIV-infectivity by interruption of the initialinteraction of the virus with the cell; this would, therefore, likelyinvolve a direct interaction of cyanovirin-N with the viral gp120. Thiswas confirmed by ultrafiltration experiments and dot-blot assays.

Ultrafiltration experiments were performed to determine if soluble gp120and cyanovirin-N could bind directly, as assessed by inhibition ofpassage of cyanovirin-N through a 50 kDa-cutoff ultrafilter. Solutionsof cyanovirin (30 μg) in PBS were treated with various concentrations ofgp120 for 1 hr at 37° C., then filtered through a 50 kda-cutoffcentrifugal ultrafilter (Amicon). After washing 3 times with PBS,filtrates were desalted with 3 kDa ultrafilters; retentates werelyophilized, reconstituted in 100 μl H₂O, and assayed for anti-HIVactivity.

FIG. 8A is a graph of OD (450 nm) versus cyanovirin concentration(μg/ml), which illustrates cyanovirin/gp120 interactions defining gp120as a principal molecular target of cyanovirin. Free cyanovirin-N wasreadily eluted, as evidenced by complete recovery of cyanovirin-Nbioactivity in the filtrate. In contrast, filtrates from cyanovirin-Nsolutions treated with gp120 revealed a concentration-dependent loss offiltrate bioactivity; moreover, the 50 kDa filter retentates were allinactive, indicating that cyanovirin-N and soluble gp120 interacteddirectly to form a complex incapable of binding to gp120 of intactvirus.

There was further evidence of a direct interaction of cyanovirin-N andgp120 in a PVDF membrane dot-blot assay. A PVDF membrane was spottedwith 5 μg CD4 (CD), 10 μg aprotinin (AP), 10 μg bovine globulin (BG),and decreasing amounts of cyanovirin: 6 μg [1], 3 μg [2], 1.5 μg [3],0.75 μg [4], 0.38 μg [5],0.19 μg [6], 0.09 μg [7], and 0.05 μg [8], thenwashed with PBST and visualized per the manufacturer's recommendations.FIG. 8B is a dot blot of binding of cyanovirin and a gp120-HRP conjugate(Invitrogen), which shows that cyanovirin-N specifically bound ahorseradish peroxidase conjugate of gp120 (gp120-HRP) in a concentrationdependent manner.

Example 7

This example further illustrates the extraordinarily broad range ofantiretroviral activity against diverse lab-adapted and clinical strainsof human and nonhuman primate immunodeficiency retroviruses.

Table 1 below shows the comparative ranges of anti-immunodeficiencyvirus activities of cyanovirin-N and sCD4 tested against a wide range ofvirus strains in diverse host cells. Particularly noteworthy is thesimilar potency of cyanovirin-N against both lab-adapted strains as wellas clinical isolates of HIV. This was in sharp contrast to the lack ofactivity of sCD4 against the clinical isolates.

The EC₅₀ values (Table 1) were determined from concentration-responsecurves from eight dilutions of the test agents (averages from triplicatewells per concentration); G910-6 is an AZT-resistant strain; A17 is apyridinone-resistant strain; HIV-1 Ba-L was tested in human peripheralblood macrophage (PBM) cultures by supernatant reverse transcriptaseactivity; all other assays employed XTT-tetrazolium (Gulakowski et al.,1991, supra). Further details of virus strains, cell lines, clinicalisolates, and assay procedures are published (Buckheit et al., AIDS Res.Hum. Retrovir. 10, 1497-1506, 1994; Buckheit et al., Antiviral Res. 25,43-56, 1994; and references contained therein). In Table 1, N.D.=notdetermined.

TABLE 1 Comparative Ranges of Antiviral Activity of CV-N and sCD4EC₅₀(nM)^(a) Virus Target Cells Cyanovirin-N sCD4 HIV-1 LaboratoryStrains RF CEM-SS 0.5 0.8 RF U937 0.5 0.1 IIIB CEM-SS 0.4 1.6 IIIB MT-20.4 13 MN MT-2 2.3 N.D. G-910-6 MT-2 5.8 N.D. A17 MT-2 0.8 13 HIV-1Promonocytotropic Isolates 214 CEM-SS 0.4 N.D. SK1 CEM-SS 4.8 N.D. HIV-1Lymphotropic Isolates 205 CEM-SS 0.8 N.D. G1 CEM-SS 0.9 N.D. HIV-1Clinical Isolates WEJO PBL 6.7 >100 VIHU PBL 5.5 >100 BAKI PBL 1.5 >100WOMEPBL 4.3 >100 HIV-2 ROD CEM-SS 7.6 >200 MS CEM-SS 2.3 N.D. SIVDelta_(B670) 174 × CEM 11 3.0

The inactivating activity of cyanovirin is conserved across anextraordinarily wide range of strains and isolates of HIV. Therefore,the epitope(s) of gp120 and/or other envelope component(s) of HIV thatare uniquely targeted by cyanovirin must be highly conserved acrossdiverse strains and isolates of HIV.

Example 8

This example further illustrates the construction of a conjugate DNAcoding sequence, and expression thereof, to provide a cyanovirin-toxinprotein conjugate that selectively targets and kills HIV-infected cells.More specifically, this example illustrates construction and expressionof a conjugate DNA coding sequence for a cyanovirin/Pseudomonas-exotoxinwhich selectively kills viral gp120-expressing host cells.

A DNA sequence (SEQ ID NO:3) coding for FLAG-cyanovirin-N and a DNAsequence coding for the PE38 fragment of Pseudomonas exotoxin (Kreitmanet al., Blood 83, 426-434, 1994) were combined in the pFLAG-1 expressionvector. The PE38 coding sequence was excised from a plasmid, adapted,and ligated to the C-terminal position of the FLAG-cyanovirin-N codingsequence using standard recombinant DNA procedures. This construct isillustrated schematically in FIG. 9. Transformation of E. coli with thisconstruct, selection of clones, and induction of gene expression withIPTG resulted in production of a conjugate protein with the expectedmolecular weight and immunoreactivity on western-blot analysis using ananti-FLAG antibody. The chimeric molecule was purified by FLAG-affinitychromatography (e.g., as in Example 4) and evaluated for toxicity tohuman lymphoblastoid cells infected with HIV (H9/IIIB cells) as well astheir uninfected counterparts (H9 and CEM-SS cells). Cells were platedin 96-well microtitre plates and exposed to various concentrations ofthe conjugate protein (named PPE). After three days, viability wasassessed using the XTT assay (Gulakowski et al., 1991, supra). FIG. 10illustrates the results of this testing. As anticipated, the infectedH9/IIIB cells expressing cell-surface gp120 were dramatically moresensitive to the toxic effects of PPE than were the uninfected H9 orCEM-SS cells. The IC50 values determined from the concentration-effectcurves were 0.014 nM for H9/IIIB compared to 0.48 and 0.42 nM for H9 andCEM-SS, respectively.

Example 9

This example illustrates transformation of a mammalian cell to express acyanovirin therein. A genetic construct suitable for demonstration ofexpression of a cyanovirin in mammalian cells was prepared by ligating aDNA sequence coding for FLAG-cyanovirin-N into the pFLAG CMV-1expression vector (IBI-Kodak, Rochester, N.Y.). The FLAG-cyanovirin-Ncoding sequence (SEQ ID NO:3) was excised from a previously constructedplasmid and ligated to the pFLAG CMV-1 vector using standard recombinantDNA procedures. African green monkey cells (COS-7 cells, obtained fromthe American Type Culture Collection, Rockville, Md.) were transformedby exposure to the construct in DEAE dextran solution. To assessexpression of FLAG-cyanovirin-N, cells were lysed after 72 hours andsubjected to PAGE and western-blot analysis. As illustrated in FIG. 11,anti-FLAG immunoreactive material was readily detected in transformedCOS-7 cells, albeit at an apparent molecular weight substantiallygreater than native recombinant FLAG-cyanovirin-N produced in E. coli.Diagnostic analyses of digests, performed in the same manner as inExample 10 which follows, indicated that this increased molecular weightwas due to post-translational modification (N-linked oligosaccharides)of the FLAG-cyanovirin-N.

Example 10

This example illustrates transformation and expression of a cyanovirinin a non-mammalian cell, more specifically a yeast cell.

A genetic construct suitable for demonstration of expression of acyanovirin in Pichia pastoris was prepared by ligating a DNA sequencecoding for cyanovirin-N into the pPIC9 expression vector (InvitrogenCorporation, San Diego, Calif.). The cyanovirin-N coding sequence (SEQID NO:1) was excised from a previously constructed plasmid and ligatedto the vector using standard recombinant DNA procedures. Yeast cellswere transformed by electroporation and clones were selected forcharacterization. Several clones were found to express, and to secreteinto the culture medium, material reactive with anti-cyanovirin-Npolyclonal antibodies (see, e.g., Example 11).

Similar to the observations with the mammalian forms described inExample 9, the elevated apparent molecular weight of the yeast-derivedproduct on PAGE and western-blot analysis, suggested thatpost-translational modification of the cyanovirin-N was occurring inthis expression system.

To further define this modification, the secreted products from twoclones were digested with peptide-N4-(N-acetyl-β-glucosaminyl)asparaoine amidase. This enzyme, obtained from New England Biolabs(Beverly, Mass.), specifically cleaves oligosaccharide moieties attachedto asparagine residues. As illustrated in FIG. 12, this treatmentreduced the apparent molecular weight of the product to that equivalentto native recombinant cyanovirin-N expressed in E. coli. Inspection ofthe amino acid sequence of cyanovirin revealed a single recognitionmotif for N-linked modification (linkage to the asparagine located atposition 30).

To further establish this as the site of glycosylation, a mutation wasintroduced at this position to change the asparagine residue toglutamine (N30Q). Expression of this mutant form resulted in productionof immunoreactive material with a molecular weight consistent with thatof native recombinant FLAG-cyanovirin-N.

Example 11

This example further illustrates an antibody specifically binding to acyanovirin.

Three 2-month old New Zealand White rabbits (1.8-2.2 kg) were subjectedto an immunization protocol as follows: A total of 100 μg ofcyanovirin-N was dissolved in 100 μl of a 1:1 suspension ofphosphate-buffered saline (PBS) and Freunds incomplete adjuvant andadministered by intramuscular injection at 2 sites on each hind leg;8-16 months from the initial injection, a final boost of 50 μg ofcyanovirin-N per rabbit was dissolved in 1000 μl of a 1:1 suspension ofPBS and Freunds incomplete adjuvant and administered by intraperitonealinjection; on days 42, 70, 98 and 122, 10 ml of blood was removed froman ear vein of each rabbit; 14 days after the last intraperitonealboost, the rabbits were sacrificed and bled out. The IgG fraction of theresultant immune sera from the above rabbits was isolated by protein-ASepharose affinity chromatography according to the method of Goudswaardet al. (Scand. J. Immunol. 8, 21-28, 1978). The reactivity of thispolyclonal antibody preparation for cyanovirin-N was demonstrated bywestern-blot analysis using a 1:1000 to 1:5000 dilution of the rabbitIgG fractions.

FIG. 13 further illustrates that the antibody prepared according to theaforementioned procedure is an antibody specifically binding to aprotein of the present invention. SDS-PAGE of a whole-cell lysate, fromE. coli strain DH5α engineered to produce cyanovirin-N, was carried outusing 18% polyacrylamide resolving gels and standard discontinuousbuffer systems according to Laemmeli (Nature 227, 680-685, 1970).Proteins were visualized by staining with Coomassie brilliant blue (FIG.13A). For western-blot analyses, proteins were electroeluted from theSDS-PAGE gel onto a nitrocellulose membrane. Non-specific binding siteson the membrane were blocked by washing in a 1% solution of bovine serumalbumin (BSA). The membrane was then incubated in a solution of the IgGfraction from the aforementioned rabbit anti-cyanovirin-N immune serumdiluted 1:3000 with phosphate buffered saline (PBS). Subsequently, themembrane was incubated in a secondary antibody solution containinggoat-antirabbit-peroxidase conjugate (Sigma) diluted 1:10000. The boundsecondary antibody complex was visualized by incubating the membrane ina chemiluminescence substrate and then exposing it to x-ray film (FIG.13B).

One skilled in the art additionally will appreciate that, likewise bywell-established, routine procedures (e.g., see Harlow and Lane, 1988,sura), monoclonal antibodies may be prepared using as the antigen aprotein of the present invention, and that such a resulting monoclonalantibody likewise can be shown to be an antibody specifically binding aprotein of the present invention.

All of the references cited herein are hereby incorporated in theirentireties by reference.

While this invention has been described with an emphasis upon preferredembodiments, it will be obvious to those of ordinary skill in the artthat variations of the preferred proteins, conjugates, host cells,compositions, methods, and the like can be used and that it is intendedthat the invention may be practiced otherwise than as specificallydescribed herein. Accordingly, this invention includes all modificationsencompassed within the spirit and scope of the invention as defined bythe following claims.

4 327 base pairs nucleic acid double linear DNA (genomic) unknown CDS10..312 1 CGATCGAAG CTT GGT AAA TTC TCC CAG ACC TGC TAC AAC TCC GCT ATC48 Leu Gly Lys Phe Ser Gln Thr Cys Tyr Asn Ser Ala Ile 1 5 10 CAG GGTTCC GTT CTG ACC TCC ACC TGC GAA CGT ACC AAC GGT GGT TAC 96 Gln Gly SerVal Leu Thr Ser Thr Cys Glu Arg Thr Asn Gly Gly Tyr 15 20 25 AAC ACC TCCTCC ATC GAC CTG AAC TCC GTT ATC GAA AAC GTT GAC GGT 144 Asn Thr Ser SerIle Asp Leu Asn Ser Val Ile Glu Asn Val Asp Gly 30 35 40 45 TCC CTG AAATGG CAG CCG TCC AAC TTC ATC GAA ACC TGC CGT AAC ACC 192 Ser Leu Lys TrpGln Pro Ser Asn Phe Ile Glu Thr Cys Arg Asn Thr 50 55 60 CAG CTG GCT GGTTCC TCC GAA CTG GCT GCT GAA TGC AAA ACC CGT GCT 240 Gln Leu Ala Gly SerSer Glu Leu Ala Ala Glu Cys Lys Thr Arg Ala 65 70 75 CAG CAG TTC GTT TCCACC AAA ATC AAC CTG GAC GAC CAC ATC GCT AAC 288 Gln Gln Phe Val Ser ThrLys Ile Asn Leu Asp Asp His Ile Ala Asn 80 85 90 ATC GAC GGT ACC CTG AAATAC GAA TAACTCGAGA TCGTA 327 Ile Asp Gly Thr Leu Lys Tyr Glu 95 100 101amino acids amino acid linear protein unknown 2 Leu Gly Lys Phe Ser GlnThr Cys Tyr Asn Ser Ala Ile Gln Gly Ser 1 5 10 15 Val Leu Thr Ser ThrCys Glu Arg Thr Asn Gly Gly Tyr Asn Thr Ser 20 25 30 Ser Ile Asp Leu AsnSer Val Ile Glu Asn Val Asp Gly Ser Leu Lys 35 40 45 Trp Gln Pro Ser AsnPhe Ile Glu Thr Cys Arg Asn Thr Gln Leu Ala 50 55 60 Gly Ser Ser Glu LeuAla Ala Glu Cys Lys Thr Arg Ala Gln Gln Phe 65 70 75 80 Val Ser Thr LysIle Asn Leu Asp Asp His Ile Ala Asn Ile Asp Gly 85 90 95 Thr Leu Lys TyrGlu 100 327 base pairs nucleic acid double linear DNA (genomic) unknownCDS 1..327 3 GAC TAC AAG GAC GAC GAT GAC AAG CTT GGT AAA TTC TCC CAG ACCTGC 48 Asp Tyr Lys Asp Asp Asp Asp Lys Leu Gly Lys Phe Ser Gln Thr Cys 15 10 15 TAC AAC TCC GCT ATC CAG GGT TCC GTT CTG ACC TCC ACC TGC GAA CGT96 Tyr Asn Ser Ala Ile Gln Gly Ser Val Leu Thr Ser Thr Cys Glu Arg 20 2530 ACC AAC GGT GGT TAC AAC ACC TCC TCC ATC GAC CTG AAC TCC GTT ATC 144Thr Asn Gly Gly Tyr Asn Thr Ser Ser Ile Asp Leu Asn Ser Val Ile 35 40 45GAA AAC GTT GAC GGT TCC CTG AAA TGG CAG CCG TCC AAC TTC ATC GAA 192 GluAsn Val Asp Gly Ser Leu Lys Trp Gln Pro Ser Asn Phe Ile Glu 50 55 60 ACCTGC CGT AAC ACC CAG CTG GCT GGT TCC TCC GAA CTG GCT GCT GAA 240 Thr CysArg Asn Thr Gln Leu Ala Gly Ser Ser Glu Leu Ala Ala Glu 65 70 75 80 TGCAAA ACC CGT GCT CAG CAG TTC GTT TCC ACC AAA ATC AAC CTG GAC 288 Cys LysThr Arg Ala Gln Gln Phe Val Ser Thr Lys Ile Asn Leu Asp 85 90 95 GAC CACATC GCT AAC ATC GAC GGT ACC CTG AAA TAC GAA 327 Asp His Ile Ala Asn IleAsp Gly Thr Leu Lys Tyr Glu 100 105 109 amino acids amino acid linearprotein unknown 4 Asp Tyr Lys Asp Asp Asp Asp Lys Leu Gly Lys Phe SerGln Thr Cys 1 5 10 15 Tyr Asn Ser Ala Ile Gln Gly Ser Val Leu Thr SerThr Cys Glu Arg 20 25 30 Thr Asn Gly Gly Tyr Asn Thr Ser Ser Ile Asp LeuAsn Ser Val Ile 35 40 45 Glu Asn Val Asp Gly Ser Leu Lys Trp Gln Pro SerAsn Phe Ile Glu 50 55 60 Thr Cys Arg Asn Thr Gln Leu Ala Gly Ser Ser GluLeu Ala Ala Glu 65 70 75 80 Cys Lys Thr Arg Ala Gln Gln Phe Val Ser ThrLys Ile Asn Leu Asp 85 90 95 Asp His Ile Ala Asn Ile Asp Gly Thr Leu LysTyr Glu 100 105

What is claimed is:
 1. An anti-cyanovirin antibody, wherein saidantibody has an internal image of gp120 of an immunodeficiency virus andcan compete with gp120 of an immunodeficiency virus for binding to acyanovirin, wherein said cyanovirin comprises SEQ ID NO:
 2. 2. Theanti-cyanovirin antibody of claim 1, wherein said immunodeficiency virusis HIV-1 or HIV-2.
 3. A method of inducing an immune response to animmunodeficiency virus in an animal, which method comprisesadministering to said animal an anti-cyanovirin antibody of claim 2 inan amount sufficient to induce in said animal an immune response to animmunodeficiency virus.
 4. A method of inducing an immune response to animmunodeficiency virus in an animal, which method comprisesadministering to said animal a cyanovirin, wherein said cyanovirincomprises SEQ ID NO: 2 and binds gp120 of an immunodeficiency virus, inall amount sufficient to induce in said animal an anti-cyanovirinantibody, wherein said amount is sufficient to induce an immune responseto an immunodeficiency virus.
 5. The method of claim 4, wherein saidanti-cyanovirin antibody can compete with gp120 of an imrmunodeficiencyvirus for binding to a cyanovirin.
 6. The method of claim 4, whereinsaid immunodeficiency virus is HIV-1 or HIV-2.
 7. A method of selectingan anti-cyanovirin antibody that has an internal image of gp120 of animmunodeficiency virus, which method comprises: (a) contacting a sampleof anti-cyanovirin antibodies with gp120 and cyanovirin, wherein saidcyanovirin comprises SEQ ID NO:2, and (b) selecting an anti-cyanovirinantibody that competes with gp120 for binding to cyanovirin, whereuponan anti-cyanovirin antibody that has an internal image of gp120 of animmunodeficiency virus is selected.
 8. The method of claim 7, whereinsaid immunodeficiency virus is HIV-1 or HIV-2.
 9. A method of selectingan anti-cyanovirin antibody that has an internal image of gp120 of animmunodeficiency virus, which method comprises: (a) contacting a sampleof anti-cyanovirin antibodies with cyanovirin and cyanovirin to which isbound gp120; and (b) selecting an anti-cyanovirin antibody that binds tocyanovirin as opposed to cyanovirin to which is bound gp120, whereuponan anti-cyanovirin antibody that has an internal image of gp120 of animmunodeficiency virus is selected.
 10. The method of claim 9, whereinsaid immunodeficiency virus is HIV-1 or HIV-2.